Folding variants of a-lactalbumin (a-la) are known to induce cell death in a number of celltypes, including mammary epithelial cells (MEC). The native conformation of a-la however hasnot been observed to exhibit this biological activity. Here we report that native bovine a-la reduced the viability of primary bovine mammary epithelial cells (BMEC) and induced caspase activity in mammospheres, which are alveolar-like structures formed by culturing primary BMECon extracellular matrix in the presence of lactogenic hormones. These observations suggest a possible role for bovine a-la in involution and/or maintaining the luminal space in mammary alveoli during lactation. In addition, co-incubation of bovine a-la in an in-vitro mammo spheremodel resulted in decreased b-casein mRNA expression and increased as1- and k-casein mRNAexpression. This differential effect on casein expression levels is unusual and raises the possibility of manipulating expression levels of individual caseins to alter dairy processing properties.Manipulation of a-la levels could be further investigated for its potential to enhance milk protein expression and/or improve lactational persistency by influencing the balance between proliferation and apoptosis of BMEC, which has a major influence on the milk-producing capacity of the mammary gland.Keywords: a-Lactalbumin, casein, apoptosis, mammary epithelial cell.Mammary epithelial cells (MEC) proliferate and differentiate during mammary gland development to form the central component of secretory alveoli. Alveoli contain a hollow sphere of MEC with apical-basal polarity, which secrete milk into the central lumen. Lumen formation and maintenance throughout lactation is achieved by a balance between MEC proliferation and apoptosis (Debnath et al.2002). The milk-producing capacity of the mammary gland is determined by the MEC population and its biosynthetic capacity;thus the balance between the rates of proliferation and apoptosis impacts on milk production and lactational persistency in dairy cattle. After peak lactation the secretory MEC population is reduced in parallel with a decrease in milk production (Boutinaud et al. 2004).Milk accumulation at weaning triggers involution of the mammary gland, which is associated with an increase in the rate of apoptosis in a proportion of the remaining MEC(Wilde et al. 1997). Since secretory MEC are in direct contact with milk, it is conceivable that milk contains pro-apoptotic factors that are involved in MEC death. It has been postulated that an autocrine suppressor of milk secretion,FIL, is present in milk (Wilde et al. 1995).A potential pro-apoptotic protein in milk is a-lactalbumin(a-la) which has been demonstrated to enhance apoptosis under certain conditions. A molten globule form of humana-la, complexed to oleic acid, termed HAMLET (humana-lactalbumin made lethal to tumour cells) induces apoptotic activity in a number of tumour cell lines (Hallgrenet al. 2006) and also in mouse MEC (Baltzer et al. 2004).HAMLET-like species derived from bovine (BAMLET),equine and porcine a-la also reduce tumour cell viability;however, active HAMLET has been found naturally only in human milk (Pettersson et al. 2006). HAMLET acts in two ways: it binds to histones in the nucleus, disrupting chromatin structures ; and it interacts with mitochondria in the cytoplasm, causing the release of cytochrome c and subsequent caspase activation (Baltzer et al. 2004). Studies in vitro indicate that a-la can bind histones without oleicacid bound to it (Permyakov et al. 2004). However, other studies suggest that complexing to oleic acid is necessary for transport into the cell (Svensson et al. 1999).