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To evaluate the effect of sequence variation within the gp120 V3 loop on CD4 T-cell recognition.CD4 T-cell clones were generated using synthetic peptides to circumvent the difficulties of using polyclonal T-cell responses. Peptides based on other HIV isolates were then used to determine the influence of single and multiple sequence differences.Three of the panels of T-lymphocyte clones (TLC), which were all specific to diverse HIV-1 clade B gp120 V3-loop peptides differing in a limited number of residues, had heterogeneous patterns of response to peptides differing in length and sequence indicating that they recognized distinct but overlapping epitopes. The panels of TLC also differed in the extent to which they tolerated sequence differences between cell-culture-adapted or primary HIV-1 isolates. One panel responded to peptides based on several clade B and one clade D isolate. In contrast, two panels, generated from two different donors using the same peptide, only responded to a limited number of clade B isolates, whereas another only recognized HIV-1BRU. Two of the panels were also stimulated by peptides based on clinical isolates from one patient with some sequence changes enhancing T-cell recognition.These data are consistent with highly diverse CD4 T-cell recognition of the HIV-1 gp120 V3 loop, which is influenced by the sequence differences within the T-cell epitopic region and has implications for the pathogenesis and design of vaccines against HIV-1.