Cell surface glycosaminoglycans (GAGs), in particular heparan sulfate (HS), have been proposed to mediate the attachment of human immunodeficiency virus type 1 (HIV-1) to target cells prior to virus entry, and both the viral gp120 envelope protein and virion-associated cyclophilin A (CypA) have been shown to directly interact with HS and its analogues. To determine the role of GAGs in HIV attachment and infection, we generated HIV-susceptible derivatives of CHO cell lines that either express high levels of GAGs (CHO-K1) or lack GAGs (pgsA745). Using a panel of HIV-1 envelopes, we found that cell surface GAG-mediated effects on virion attachment and infection vary in an envelope strain-dependent but coreceptor-independent manner. In fact, cell surface GAG-mediated enhancement of infection is confined to isolates that contain a highly positively charged V3-loop sequence, while infection by most strains is apparently inhibited by the presence of GAGs. Moreover, the enhancing and inhibitory effects of polycations and polyanions on HIV-1 infection are largely dependent on the presence of cell surface GAGs. These observations are consistent with a model in which GAGs influence in vitro HIV-1 infection primarily by modifying the charge characteristics of the target cell surface. Finally, the effects of GAGs on HIV-1 infection are observed to an equivalent extent whether CypA is present in or absent from virions. Overall, these data exclude a major role for GAGs in mediating the attachment of many HIV-1 strains to target cells via interactions with virion-associated gp120 or CypA.