Mammary epithelial cell cultures from transgenic mice carrying the human serum albumin (HSA) gene or minigenes behind the regulatory sequences of the ovine beta-lactoglobulin gene were analyzed. Previously, we demonstrated that non-HSA-secreting transgenic strains retain the potential to express the HSA transgene in vitro and that mammary epithelial cell cultures from non-HSA-secreting strains express higher levels of HSA when grown on tissue culture plastic than they do when grown on collagen. In this study we studied the expression of BLG/HSA fusion genes in epithelial cell cultures of additional transgenic strains and additional substrata. Our results show that: (1) The BLG/HSA fusion gene in only one of seven HSA-secreting or nonsecreting transgenic strains tested accurately responded to signals from the EHS matrix; (2) HSA DNA sequences dominantly affected the activity of BLG as well as the whey acidic protein promoters; and (3) HGF/SF induced both milk proteins and HSA gene expression. These results suggest that the response to the extra cellular matrix (ECM) plays a key role in the expression of BLG/HSA fusion genes and that the function of the regulatory elements within the promoter regions of milk protein genes involved in response to the ECM, in developmental and in tissue specificity, very much depend on the downstream gene sequences.