Cell surface glycosaminoglycans (GAGs) are responsible for the majority of respiratory syncytial virus (RSV) attachment to cultured cells leading to infection. The viral glycoprotein G binds to GAGs and was thought to be the viral attachment protein, but more recently virus lacking the G protein was shown to be infectious in cell culture. We have compared the GAG dependence of a recombinant, green fluorescent protein-expressing virus containing the F protein as its only viral glycoprotein (rgRSV-F) to isogenic complete virus containing all three viral glycoproteins (rgRSV-SGF). Attachment and infection by each virus was found to be largely dependent on cell surface heparan sulfate (HS) based on the finding that both activities were inhibited by preincubation of virus with soluble HS, by removal of HS from target cells by enzymatic treatment or mutation, or by pretreatment of the target cells with basic fibroblast growth factor (bFGF), which binds HS. These results, coupled with the previous finding that SH is not involved in virion binding (S. Techaarpornkul, N. Barretto, and M. Peeples, 2001, J. Virol. 75, 6825-6834), suggest that, in the context of the virion, both the G and F proteins bind to HS. Interestingly, both rgRSV-F and rgRSV-SGF retained significant binding activity and infectivity despite these treatments, suggesting an alternate productive attachment and infection pathway. This property of GAG independence was particularly apparent for rgRSV-F virions, which retained nearly half of its attachment and infection activities in most of these experiments. Comparison of the attachment and infection activities of rgRSV-SGF and rgRSV-F virions with a Chinese hamster ovary cell line and a derivative thereof that is defective in GAG synthesis indicated that approximately 50% of rgRSV-SGF attachment is due to G protein-GAG binding, 25% to F protein-GAG binding, and 25% to an independent pathway. This alternative pathway presumably is mediated by the sole remaining viral surface protein, F, although the formal possibility exists that some other virion-associated protein is involved.