During lactation the mammary gland synthesizes a large amount of glycoproteins including those composing milk fat globule membrane (MFGM). Our previous study showed that N-linked sugar chains with GalNAc beta1-->4GlcNAc structure appears to increase in bovine MFGM glycoproteins during early lactation [Ujita et al. (1993) FEBS Lett. 332, 119-122]. Western blot analysis of membrane glycoproteins from lactating and post-lactating bovine mammary glands using Wistaria floribunda agglutinin (WFA), which binds oligosaccharides terminating with beta-N-acetylgalactosamine, and Ricinus communis agglutinin-I (RCA-I), which binds oligosaccharides preferentially terminating with beta-1,4-galactose, showed that the number and reactivity of protein bands to WFA but not to RCA-I decrease drastically in the post-lactating mammary sample. Establishment of primary cultured epithelial cells from lactating bovine mammary gland and their culture on collagen-coated dishes in the presence of a mixture of lactogenic hormones revealed that N-linked sugar chains with GalNAc beta1-->4GlcNAc structure are expressed in the functionally differentiated cells without altering the apparent beta-galactosylation of the oligosaccharides. These results strongly suggest that the expression of GalNAc beta1-->4GlcNAc structure on N-linked sugar chains is associated with the mammary gland differentiation.