The functions of the surface glycoproteins (SU) of feline leukemia viruses (FeLVs) are of interest since these proteins mediate virus infection and interference and are critical determinants of disease specificity. In this study, we examined the biochemical and genetic determinants of SU important to virus entry and cell killing. In particular, we developed and used vesicular stomatitis virus (VSV)/FeLV pseudotype virus interference assays to determine interference subgroupings and assess mechanisms of host cell restriction. We also assessed roles of SU in virus growth kinetics and in the inhibition of cell killing caused by superinfection with cytopathic virus. Subgroup classification by VSV/FeLV pseudotype assay was in agreement with that defined previously by focus interference assay and was found to be determined by changes near the N terminus of SU for FeLV subgroups A (FeLV-A) and C. Virus host range restriction was found to be mediated at the level of virus entry in most cases, although postentry events mediated restriction in the failure of a subgroup A-like, T-cell cytopathic and immunodeficiency-inducing clone (FeLV-FAIDS-EECC) to replicate in feline fibroblasts. FeLV-FAIDS-EECC-induced cell killing was also inhibited by prior infection with one of two FeLV-A isolates. This inhibition could be conveyed by as few as four amino acid changes near the N terminus of the FeLV-A SU and also appeared to be mediated at a postentry level. Lastly, the SU-coding sequence was also found to determine differences in growth kinetics of viruses within the same subgroup. These studies demonstrate that subtle alterations in the FeLV SU, particularly in the N-terminal region, impart multiple significant functional differences which distinguish virus variants.