The function of microtubules in plant cells relies on their interactions with various, largely unidentified, proteins. A 75-kDa polypeptide (p75) was isolated previously by tubulin affinity chromatography of tobacco BY-2 proteins and is further characterized here. We have obtained two peptide sequences of 13 and 8 amino acid residues from the p75, which have 77% and 87% similarity to two putative dTDP-glucose 4,6-dehydratase proteins of 74.9 and 75.3 kDa in Arabidopsis. The proteins contain a peroxisomal matrix targeting signal SKL or AKL near their carboxyl terminus, an epimerase-dehydratase motif, and a region of 321 amino acid residues sharing 41% similarity with a dTDP-glucose 4,6-dehydratase from Aneurinibacillus thermoaerophilus. A monoclonal antibody 4B9 against the tobacco p75 reacted with a 75-kDa polypeptide on immunoblots of tobacco BY-2 and Arabidopsis protein extracts, and in immunofluorescence microscopy it revealed small organelle-like structures in tobacco BY-2 and Arabidopsis root-tip cells. Double labelling with an antibody against the peroxisomal marker enzyme, catalase, showed that the organelles are indeed peroxisomes. The peroxisomes were in closer association with actin filaments than microtubules. This observation supports recent findings that plant peroxisomes move on actin filaments. We propose that the peroxisomal dTDP-glucose 4,6-dehydratases may be involved in plant defence responses to oxidative stress.