Proteoglycans (PGs) were extracted from the [35S]-sulfate labelled medium and cell layer of proliferating human gingival epithelial cells and analyzed by ion exchange and molecular sieve chromatography, and by SDS-PAGE. The majority of the incorporated radioactivity secreted into the medium eluted from a DEAE Sephacel ion exchange column as a single peak at 0.44 M NaCl with a small shoulder at 0.52 M NaCl. This material, when chromatographed on Sepharose CL-6B contained two species--a quantitatively major peak at K(av) = 0.30 (M(r) congruent to 235,000 on SDS-PAGE) and a quantitatively minor peak at K(av) = 0.39. The major peak was sensitive to alkaline borohydride, shifting to K(av) = 0.45, and nitrous acid degradation, indicating the presence of heparan sulfate PG with glycosaminoglycan chains with M(r) congruent to 26,000. The minor peak is chondroitin/dermatan sulfate PG with glycosaminoglycan chains of M(r) = 22,200 as indicated by sensitivity to alkaline borohydride (shifting to K(av) = 0.48) and chondroitin ABC lyase digestion. The [35S]-sulfate labelled material from the cell layer eluted in a broad peak between 0-0.50 M NaCl from DEAE Sephacel. Chromatography of this material on Sepharose CL-6B revealed the presence of three peaks at K(av) = 0.20, 0.31, and 0.75. The largest peak (K(av) = 0.20 and M(r) congruent to 245,000 on SDS-PAGE) shifted elution position to K(av) = 0.50 after alkaline borohydride treatment and was completely sensitive to nitrous acid degradation. These results indicate that this peak contains heparan sulfate PG with glycosaminoglycan chains of M(r) congruent to 20,000. Two peaks containing [35S]-sulfate labelled glycosaminoglycan chains were detected by chromatography of the cell layer extract over Sepharose CL-6B with K(av)S = 0.42 (M(r) congruent to 30,500) and 0.75 (M(r) congruent to 5300). The larger peak was predominantly chondroitin/dermatan glycosaminoglycan as indicated by susceptibility to chondroitin ABC lyase while the chains at K(av) = 0.75 were predominantly heparan sulfate with 83% susceptibility to nitrous acid. These results indicate that cultured human gingival epithelial cells synthesize and secrete principally heparan sulfate PGs with small amounts of chondroitin/dermatan sulfate PGs. This work will serve as a basis for future studies designed to examine those factors involved in regulation of PG synthesis by these cells.