Bovine mammary epithelial cells were isolated and purified by using collagenase digestion of the mammary gland tissue for the primary cell culture, and subsequently using trypsin-selected digestion of the cultured cells for cell purification. Morphological observation revealed that the cultured cells possessed the typical character of epithelial cells. Karyotyping analysis showed the normal chromosome number in cultured cells. The tissue-specific expression of cytokeratin 5 and 8 genes in mammary epithelial cells was identified by immunofluorescent cytochemical staining. The transcription of the betacasein gene was detected by RT-PCR, when the purified cells were induced with insulin, hydrocortisone and prolactin in the culture medium without serum. The above results indicate that the purified cells are mammary epithelial cells, which can transcript beta-casein mRNA in the induction condition.