The mechanisms that control the synthesis of milk protein are not fully understood and could well involve undiscovered proteins within the mammary gland. A search for such proteins in high salt extracts of nuclei that had been isolated from bovine mammary tissues was undertaken using two-dimensional electrophoresis on large format gels. The sensitivity of the procedure was sufficient to detect the transcription factors Sp1 and NF-1 by Coomassie blue stain; over 300 proteins were routinely detected. Analysis of mammary tissue taken from 5 nonlactating cows in midpregnancy, 5 cows in late lactation, and 4 cows in early involution revealed five proteins where relative abundance was altered with stage of lactation or reproductive cycle. Four of these proteins were identified by Western blotting or amino acid sequencing as lactoferrin, annexin II, vimentin, and heavy-chain immunoglobulin. Analysis of proteins after further enrichment of the extracts by heparin-Sepharose affinity chromatography revealed an additional protein that was substantially more abundant in samples from lactating cows. This 90-kDa protein did not react with anti-Stat5 antibodies. Conceivably, one or more of these six proteins could play a role in the lactational function of the bovine mammary gland.