Eye formation is an important feature for various cheese varieties. Thisstudy firstly aimed to evaluate the potential of X-ray computed tomography (CT) andimage analysis software as a non-invasive method to quantify cheese eye volume.The quantification of the eye volume by CT was validated with 12 eyeless hardcheeses made with the inclusion of 0'100 hollow PP balls (Ãƒ'=10 or 20 mm). Theresults obtained for the total volume of the 'artificial eyes' showed a good correlationwith the volume of the added balls (R2>0.998). In a second part of the study, thedeveloped CT method was applied for the non-invasive investigation of the eyeformation in semi-hard Tilsit-type cheese with adjuncts of Lactobacillus casei orLactobacillus plantarum. Both adjuncts completely metabolized citrate in cheese,thus enhancing gas production. Total eye number, relative eye volume and the sizedistribution of eyes were determined in order to reveal the influence of the twoadjunct cultures on eye formation. In comparison to the control, the addition of L.casei resulted in a significantly higher eye number and eye volume. In contrast, theaddition of L. plantarum did not influence the eye formation in a significant way,though it yielded cheeses with more succinate and aspartate and less serine atsignificant levels. The results imply that, apart from CO2 production, other factors,such as the presence of eye nuclei and the dynamics of CO2 diffusion, interfere in animportant way in the process of eye formation in cheese.