The substitution of glycine for valine at amino acid 448 in the C4 domain of SIVmac239 envelope glycoprotein gp120 resulted in little or no viral infectivity for CEMX174 cells and rhesus monkey peripheral blood mononuclear cells. The block to viral replication was located at an early stage including virus entry into cells since the mutant virus was severely impaired in its ability to form newly synthesized viral DNA during the initial 14 hr after exposure to cells. Envelope glycoprotein with the 448V-->G mutation bound to soluble CD4 in a manner similar to wild type in an in vitro assay and was also translated, processed, and cleaved in a manner similar to wild-type envelope protein. When CEMx174 cells were transfected with 448V-->G mutant proviral DNA and held for longer than 40 days, a variant appeared in one culture that was able to replicate with near wild-type kinetics. Sequencing of viral DNA from cultures infected with this variant revealed that the original 448V-->G mutation was retained in eight of eight clones analyzed but that six of eight clones analyzed had an additional mutation of V-->I at amino acid 322 of gp120. Amino acid 322 in SIVmac gp120 is located in a region which corresponds to V3 of HIV. Analysis of site-specific mutants demonstrated that the mutation of V-->I at position 322 can indeed compensate for the 448V-->G mutation, resulting in efficient virus entry and replication by the double mutant. These results identify single amino acids in gp120 that are critical for early events in SIVmac replication and suggest that the "V3" and C4 domains of SIVmac gp120 cooperate to effect virus entry.