The objective of these studies was to develop serum-free culture conditions for dissociated acini from rat submandibular glands. Acini were isolated from the submandibular glands of 42-46 d old rats and cultured on reconstituted rat tail collagen containing laminin in 1:1 Ham's F12 and Dulbecco's media, supplemented with BSA, transferrin, insulin, T3, EGF, dexamethasone, retinoic acid, carbamylcholine, and trace elements, and gassed with 50% O2. The acini became partly embedded in the collagen gel and rapidly enlarged throughout the first 22 d of culture, maintaining modest seromucous acinar differentiation, as judged morphologically and by mucin secretion. Parallel cultures then were grown under 20, 35, 50, and 65% O2, and evaluated morphologically and by DNA content. Growth and retention of seromucous acinar characteristics were best with 35% O2, but lipid accumulation and cell death were unacceptably high. A spectrum of concentrations of insulin and glucagon then were tried. With 0.05 micrograms/ml insulin, cellular growth and organization were orderly, lipid accumulations were not excessive, and moderate differentiation was retained through 15 d of culture. With more than 0.1 microgram/ml insulin added to or subtracted from the optimum, the detrimental effects recurred. Addition of sufficient glucagon counteracted the effects of both optimum and excessive concentrations of insulin. We now have achieved an orderly growth of moderately differentiated rat submandibular acini for 15 d in serum-free primary culture.