The use of rat testicular seminiferous tubular basement membrane (STBM) segments as a model substratum for the in vitro maintenance of tumor cells dissociated from a transplantable pancreatic acinar rat carcinoma (J. K. Reddy and M. S. Rao, Science (Wash. DC), 198: 78-80, 1977) is described. Ultrastructurally pure, hollow tubular segments of STBM were prepared by mechanical disaggregation, DNase digestion, and deoxycholate treatment. Dissociated pancreatic acinar carcinoma cells adhered readily to STBM segments within 1 to 6 hr, and these STBM-tumor cell aggregates were maintained for up to 7 days in serum-free chemically defined medium supplemented with hydrocortisone, insulin, vitamin C, and soybean trypsin inhibitor. The tumor cells formed acinar-like clusters and displayed intercellular junctions and polarization of secretory granules toward the center of these clusters. By 4 days, virtually all cells of this acinar carcinoma maintained on STBM in supplemented chemically defined medium contained numerous secretory granules. Cell replication, as determined by [3H]thymidine autoradiography, ceased within 18 hr of attachment of neoplastic cells to STBM; however, all cells incorporated [3H]leucine as evidenced by light and electron microscopic autoradiography. In addition, two-dimensional analysis and fluorography of newly synthesized secretory proteins discharged by these cells in response to carbamylcholine revealed the presence of Mr 24,000 protein and 19 other secretory proteins characteristic of this tumor (L. J. Hansen, M. K. Reddy, and J. K. Reddy, Proc. Natl. Acad. Sci. USA, 80: 4379-4383, 1983). The culture system utilizing STBM and supplemented chemically defined medium should allow investigation of the effects of a variety of factors on morphogenesis, cytodifferentiation, and gene expression in pancreatic acinar tumors.