Mouse mammary epithelial cells (MMEC) secrete certain milk proteins only when cultured on floating collagen gels. We demonstrate here that modulation of milk proteins by substrata is manifested at several regulatory levels; (i) Cells cultured on floating collagen gels have 3- to 10-fold more casein mRNA than cells cultured on plastic or attached collagen gels. (ii) Cells on the latter two "flat" substrata, nevertheless, synthesize a significant amount of caseins, indicating that the remaining mRNA is functional. (iii) Cells on all substrata are inducible for casein mRNA and casein proteins by prolactin, but the extent of induction is greater on collagen than that on plastic--i.e., the substratum confers an altered degree of inducibility. (iv) Cells on all substrata synthesize casein proteins at rates proportional to the amount of casein mRNA, but the newly synthesized caseins in cells on plastic are degraded intracellularly, whereas those synthesized by cells on floating gels are secreted into the medium. (v) Cells on all substrata examined lose virtually all mRNA for whey acidic protein despite the fact that this mRNA is abundant in the mammary gland itself; we conclude that additional, as-yet-unknown, factors are necessary for synthesis and secretion of whey acidic protein in culture.