We have previously shown that medium conditioned by virus producer cells inhibits retrovirus transduction, and that a portion of the inhibitory activity is sensitive to chondroitinase ABC. In this study, we have quantitatively evaluated the fraction of the inhibitory activity that is due to chondroitinase ABC-sensitive material and partially characterized the inhibitors. The kinetics of chondroitinase ABC digestion of glycosaminoglycans and virus inhibitory activity in cell culture medium were measured, and the results used to estimate the amount of the chondroitinase ABC-sensitive virus inhibitory activity that was initially in the medium. We found that up to 76% of the inhibitory activity of medium conditioned by packaging cells derived from NIH 3T3 cells is sensitive to chondroitinase ABC. The remainder of the inhibitory activity is not sensitive to other glycosaminoglycan lyases (heparitinase I or heparinase I), which suggests that substances other than glycosaminoglycans or proteoglycans are present in virus stocks and inhibit transduction. To further characterize the inhibitors, proteoglycans from conditioned medium were purified by batch anion exchange and size exclusion chromatography. Two major size groups (100 kDa and 950 kDa) of proteoglycans were isolated. Transduction was inhibited 50% by 0.6 mu g/mL of the high-molecular-weight proteoglycan or by 1.7 mu g/mL of the low-molecular-weight proteoglycan. Significantly, the proteoglycans, because of their large size and poor sieving properties, coconcentrated with virus particles concentrated by ultrafiltration and prevented any significant increases in transduction efficiency. Transduction efficiencies of virus stocks were increased more than tenfold by ultrafiltration, but only when the concentrated virus was treated with chondroitinase ABC.