Both Sertoli and myoid cells have been shown to be required for the appropriate deposition of basement membrane in the testis. We sought to define the pattern of basement membrane gene expression in Sertoli and peritubular myoid cells in vitro in order to begin to understand the regulatory mechanisms involved in basement membrane synthesis. Sertoli and myoid cells cultured alone or together were examined for synthesis of basement membrane components. Immunocytochemical localization demonstrated that Sertoli cells alone produced laminin and collagen IV, but not fibronectin, while myoid cells produced all three proteins. In Sertoli:myoid cocultures, a sequential deposition of the components into extracellular fibers was noted during 5 days of culture. Northern blot analysis revealed that mRNA levels for the laminin B1 chain and collagen IV increased from Days 3 to 5 in Sertoli cell monocultures. By contrast, the levels of laminin B1, collagen IV, heparan sulfate proteoglycan, and fibronectin decreased in the cocultures. Transcripts for the laminin A chain were not detected in the myoid cells; instead these cells produced the mRNA for the laminin homologue, merosin. This observation was confirmed by immunolocalization of merosin to the tunica propria of the testis and in cultured myoid cells. These data describe the expression of the basement membrane genes by Sertoli and peritubular myoid cells and provide the basis for future studies to determine the mechanisms that regulate the expression of the basement membrane genes in the testis.