A 35SO4-labeling/chromatography technique has been developed which facilitates quantitation of sulfated glycosaminoglycan (GAG) synthesis in mammalian cell cultures. The technique has been used to compare sulfated GAG biosynthesis, degradation, and turnover in three related cell lines with differing degrees of density-dependent inhibition of growth in vitro (Balb/c 3T3, SV3T3, and SV3T3 revertant cells). Viral transformation of Balb 3T3 cells is accompanied by a 2-5-fold decrease in cell associated sulfated GAG. SV3T3 revertant cells, which show partial reversion to low saturation density in vitro, show a 2.5-8-fold increase in cell-associated sulfated GAG compared to the parental SV3T3 cells from which they were selected. In addition, the distribution of 35SO4 and [3H]glucosamine among the different GAG species produced by SV3T3 revertant cells reverts so that it is similar to the distribution characteristic of untransformed 3T3 cells rather than SV3T3 cells. Mild trypsin treatment of 35SO4-labeled cells removed 68-84% of the cellular sulfated GAG, suggesting that at least this proportion of the total cellular sulfated GAG was located at the cell periphery. Removal of 35SO4-labeled cells from the Petri dish with a Ca2+ selective chelating agent revealed a fraction of the sulfated GAG that remained tightly bound to the Petri dish. A higher proportion of the total cell-associated sulfated GAG remained attached to the Petri dish in cultures of untransformed and revertant cells compared to that present in cultures of transformed cells. A role for sulfated GAG in density-dependent growth inhibition of fibroblast cultures is proposed and discussed in the light of the data obtained.