The surface glycoproteins of viruses can play important roles in viral attachment, entry, and morphogenesis. Here, we investigated the role of the attachment G glycoprotein of human respiratory syncytial virus (RSV) in viral infection. RSV G is produced both as a complete, transmembrane form and as an N-terminally truncated form that is secreted. Using reverse genetics, we created mutant recombinant RSVs (rRSV) that do not express G (DeltaG) or express either the secreted or the membrane-bound form of G only (sG and mG, respectively). In Vero cells, the DeltaG virus formed plaques and grew as efficiently as wild-type rRSV and mG. In contrast, DeltaG replicated less efficiently and did not form distinct plaques in HEp-2 cells. This defect was primarily at the level of the initiation of infection, with only a minor additional effect at the level of packaging. Replication of DeltaG in the respiratory tract of mice was very highly restricted, indicating that G is important in vivo. Although the G protein expressed by the sG virus was confirmed to be secreted, this virus grew at least as efficiently as wild-type in HEp-2 cells and was only moderately attenuated in vivo. Thus, the G protein was important for efficient replication in HEp-2 cells and in vivo, but this function could be supplied in large part by the secreted form and thus does not require the cytoplasmic and transmembrane domains. Amino acids 184-198 have been identified as the major heparin-binding domain of the G protein and were implicated in mediating binding to cells [S. A. Feldman et al., 1999, J. Virol. 73, 6610-6617]. Heparin-like glycosaminoglycans also appeared to be important for infection in vitro by direct clinical isolates of RSV. Deletion of amino acids 187-197 from rRSV did not reduce its sensitivity to neutralization in vitro by incubation with soluble heparin, did not reduce its efficiency of growth in vitro, and resulted in only a modest reduction in vivo. Thus, the putative heparin-binding domain is not the sole determinant of heparin sensitivity and is not a critical functional domain.