We isolated 59 Chinese hamster ovary cell mutants defective in 35SO4 incorporation into glycosaminoglycans. Thirty-five mutants incorporated [6-3H]glucosamine into glycosaminoglycans normally, suggesting that they were specifically impaired in sulfate incorporation. Cell hybridization studies revealed that the 35 mutants defined a unique complementation group. Pulse-labeling one of the mutants with 35SO4 showed that it possessed a defect in a saturable, 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid-sensitive transport system required for sulfate uptake. Despite the dramatic reduction in 35SO4 incorporation, the mutant synthesized sulfated heparin and chondroitin chains. Incubation of the mutant with [35S]cysteine resulted in the formation of 35SO4, which was subsequently incorporated into the glycosaminoglycans. Similar results were obtained when wild-type cells were incubated in sulfate-free growth medium containing [35S]cysteine, and isotope dilution analysis indicated that about 15 μM of sulfate was derived from cysteine catabolism. We also found that the sulfate transport deficiency rendered the mutant resistant to 5 μM sodium chromate, whereas wild-type cells did not divide under these conditions. However, the mutant also did not proliferate in medium containing 5 μM chromate when grown in the presence of wild-type cells, suggesting that chromate was transported through cell-cell contacts. Since co-cultivating sulfate transport-deficient mutants with mutants defective in xylosyltransferase or galactosyltransferase I partially restored 35SO4 incorporation into glycosaminoglycans, intercellular sulfate transport occurred as well. Therefore, the availability of sulfate for glycosaminoglycan synthesis depends on sulfate uptake, turnover of sulfur-containing amino acids, and sulfate transport between cells.