A T-cell-line-tropic, syncytium-inducing, sCD4- and serum neutralization-sensitive variant (R3H) of the macrophage-tropic, non-syncytium-inducing, sCD4- and serum neutralization-resistant molecular clone HIV-1SF162 was obtained by passage through the T-cell line HUT 78. Sequence analyses of the V1-V5 regions of envelope gp120 of the variant R3H revealed amino acid substitutions in the V3 (amino acids 307, 312) and V4 (amino acid 390) domains. Site-directed mutagenesis of the HIV-1SF162 genome showed that specific mutations in the V3 loop of this isolate can alter tropism and cytopathicity of the virus, but only moderately affect its sensitivity to sCD4 and serum neutralization. These results show that adaptation to growth in T-cell lines can render a primary-like virus sensitive to sCD4 and serum neutralization. However, the extent of T-cell line tropism does not correlate with the degree of susceptibility to sCD4 and serum neutralization. The latter appears to be dependent on the amino acid compositions of the V3 loop and other regions of envelope gp120, whereas the former is primarily determined by the V3 loop. Our findings further illustrate the importance of the V3 loop in influencing HIV-1 cell tropism and syncytium formation, and the interplay among V3 loop residues in maintaining a structure of the loop that influences biological phenotype of the virus.