Uteroglobin (UG) is a secretory protein produced by the rabbit endometrium and its production is increased during cell differentiation which occurs during early pregnancy or pseudopregnancy. In the present study, the optimal conditions for UG production by rabbit endometrial epithelial cells in culture were examined. Metabolic labeling studies showed the incorporation of [35S]methionine into UG molecules by the endometrial epithelial cells in culture. Accumulation of UG in culture media was linear for at least a period of 24 h. These cells do not catabolize exogenously added radiolabeled UG. Endometrial cells obtained from virgin female rabbits at different times after the administration of human CG (hCG) and put in culture were found to make different amounts of UG. The maximal UG production was found in cells taken from pseudopregnant rabbits 4 days after hCG administration. Cycloheximide (28 micrograms/ml) inhibited the production of UG by the cells in culture whereas actinomycin-D (5 micrograms/ml) and cordycipin (50 micrograms/ml) increased its production. Inhibition of DNA synthesis by hydroxyurea (10(-3) M) did not affect the UG production. The production of UG was significantly less when cells were cultured on attached or floating collagen gels as compared to cells grown on plastic Petri dishes. The amino acid content of Ham's F-12 medium was shown to be adequate for maximal UG production; lowering this amino acid concentration decreased the amount of UG accumulated in the medium over a 24-h period. Increasint the number of cultured cells per dish resulted in an increased UG production per cell.