Several types of tumors contain high concentrations of hyaluronate, yet isolated tumor cells in culture often produce little glycosaminoglycan. To explore the possibility that interactions between tumor cells and host fibroblasts stimulate hyaluronate synthesis, human tumor cells were grown separately from and in coculture with normal human fibroblasts. Stimulation was observed with each of the three types of tumor cells used: LX-1 lung carcinoma, DAN pancreatic carcinoma, and TRIG melanoma. The interaction between LX-1 cells and fibroblasts was studied in detail. Under serum-free conditions, cocultures of LX-1 and fibroblasts synthesized 3-fold more hyaluronate than the sum of that produced by LX-1 and fibroblast cultures grown separately. This stimulation was linear over 72 hr and hyaluronate represented 80% of the glycosaminoglycan synthesized. Maximum stimulation occurred at a ratio of fibroblasts to LX-1 cells of 1-2:1. Quantitation of unlabeled glycosaminoglycans by HPLC analysis of disaccharides generated by digestion with chondroitin ABC and AC lyases (EC 4.2.2.4 and 4.2.2.5) demonstrated that net accumulation of hyaluronate increased 2-fold and that hyaluronate represented 80% of total chondroitinase-sensitive glycosaminoglycan produced by the cocultures. The disaccharide patterns obtained showed that accumulations of chondroitin-4- and chondroitin-6-sulfates were stimulated proportionately to that of hyaluronate in these cocultures. Similar levels of stimulation due to coculture were obtained in serum-containing and serum-free media. Stimulation was not effected by addition of LX-1-conditioned medium to fibroblast cultures or by culturing LX-1 and fibroblasts under conditions where they shared the same medium but were physically separated. Cell contact between LX-1 and fibroblasts thus appears to be necessary for the stimulation of hyaluronate synthesis.