The developmental and hormonal regulation of UDP-GlcNA:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme which initiates the biosynthesis of asparagine-linked glycoproteins, was investigated in mouse mammary glands. An anti-peptide antibody raised against the carboxyl-terminal 11 amino acids of mouse GPT, immunoadsorbed GPT activity and recognized a protein of expected size (approximately 48 kDa) on Western blots. Mouse mammary glands at different stages of development were examined for GPT activity, immunoreactive protein, and GPT mRNA. All three parameters showed a similar trend, i.e. they were low in tissues from virgin and pregnant animals, increased steadily during lactation, reaching a peak around mid to late lactation, and declined thereafter in glands from post-lactating animals. At mid-lactation, the increase in GPT activity, immunoreactive protein, and GPT mRNA relative to the virgin stage was 9.5-, 3.3- and 5.4-fold respectively, on a per cell basis. These data suggest possible transcriptional and post-transcriptional modulation of GPT gene expression during development of mouse mammary gland. The results on hormonal regulation of GPT in mouse mammary explants and primary mouse mammary epithelial cells showed that all three parameters cited above were stimulated maximally by the combined presence of insulin, hydrocortisone, and prolactin, indicating that the hormonal regulation of GPT expression is also mediated at the level of RNA.