We analyzed the synthesis of DNA, the rate of cell proliferation, and the expression of milk protein genes in mammary cells grown as primary cultures on or in collagen gels in chemically defined media. We assessed DNA synthesis and cell growth, measured by [aH]-thymidine incorporation into acid-insoluble material, DNA content, and cell counts, in a progesterone-and prolactin-containing medium. In some experiments, cultures were pulsed for I h with [3H]thymidine and dissociated into individual cells which were cytocentrifuged and processed for immunocytochemistry and autoradiography. We analyzed expression of milk protein genes at the transcriptional, translational, and posttranslational levels in a progesteronedepleted medium in the presence or absence of prolactin. We measured protein secretion by radioimmunoassays with antisera directed against caseins, ~-lactalbumin and milk transferrin. We determined protein synthesis by incorporating radio-labeled amino acids into acid-precipitable material and by immunoprecipitating biosynthetically labeled milk proteins. We assessed the accumulation of casein mRNA by hybridizing total cellular RNA extracted from cultured cells with 32p-labeled casein cDNA probes. On attached collagen gels, the cells synthesized DNA and replicated until they became confluent. The overall protein synthetic activity was low, and no milk proteins were synthesized or secreted even in the presence of prolactin. The block in milk protein gene expression was not restricted to translational or posttranslational events but also included transcription, since no casein mRNA accumulated in these cells. On floating gels, protein synthesis was threefold higher than in cells from attached gels. Overall protein synthesis as well as casein and ~-lactalbumin synthesis and secretion were prolactindependent with maximal stimulation at around 10 -9 M. A marked inhibition occurred at higher hormone concentrations. Casein mRNA accumulated in these cells, provided prolactin was present in the medium. In contrast, these cells did not synthesize DNA, nor did they replicate. In embedding gels, the rate of cell proliferation was exponential over 25 d with a doubling time of ~70 h. The overall protein synthesis increase was parallel in time with the increase in cell number. Caseins and ~-lactalbumin (in contrast to transferrin) were synthesized only in the presence of prolactin. We observed the same hormone dependency as with cells growing on floating gels. The number of casein-and transferrin-positive cells was measured after dissociating the cell cultures. At day 12, 60% of the total cells stored transferrin in small cytoplasmic vesicles, whereas only 25% of the cells accumulated casein. Differences in the organization and in the shape of mammary cells depending on cell surface conditions suggest that the geometry of the cells, their interaction with extracellular matrix constitutents, and cellto-cell interactions play a role in the expression of two mammary functions: DNA synthesis and growth, as well as m...