The level of proviral DNA sequence variation in the V1V2 region was monitored over time in six HIV-1-infected individuals. Substitutional and length variation was observed, where the majority of length changes, ranging from 28 to 49 amino acids, was located within the V1 region. Evidence for convergent evolution in the V2 region was found. The functional significance of this variation was assessed by cloning the V1V2 sequences into an infectious molecular clone, HXB2. The majority of chimeras replicated, demonstrating that the sequences, though genetically distinct, were capable of conferring a viable phenotype. Chimeras expressing closely related sequences in a constant genetic background displayed different biological phenotypes, with respect to both cytopathicity and cell tropism. However, no association between primary V1V2 amino acid sequence and viability or cytopathicity of the chimeric virus was observed, suggesting that predictions of virus phenotype based on sequences alone may be incorrect. The effect of V1V2 variation on the overall gp 120 conformation was measured by expressing the gp 20 from a number of viable and nonviable clones. No differences were observed, suggesting that misfolding of the chimeric gp 120 protein was not an explanation for the nonviability of some virus clones. Several chimeras were noncytopathic and only able to replicate in PBMC cultures, demonstrating that the V1V2 region, independent of the V3 sequence, is capable of defining both tropism and cytopathicity.