The biosynthesis of selenoproteins was studied in relation to milk formation and mammary cell biology by incubating the bovine mammary cell line MAC-T with ((75)Se)selenite. Intracellular proteins and proteins secreted into the cell culture medium were separated by 2D electrophoresis, the selenoproteins were detected by autoradiography, and the proteins were identified by MALDI-TOF. Approximately 35 (75)Se-containing spots were found in the cell proteins from MAC-T cells. Among them, one-third showed high intensity. The strongest spot was identified as glutathione peroxidase 1. About 20 spots were observed in protein precipitated from cell culture medium, one-third of them being distinctly visible. In an attempt to study a perturbation of the system, the effect of retinoic acid (RA) on the formation of selenoproteins was investigated. The concentration of (75)Se in total cell protein was reduced by about 35% in cells cultured with RA compared with control cells, while the opposite effect was observed in protein precipitated from cell culture medium, which contained 60% more (75)Se in RA-treated samples than in controls. There were also indications that RA might affect different selenoproteins in different ways. The methods described provide a promising approach for further studies of the regulation of selenoprotein formation in the mammary gland.