T-20 is a synthetic peptide that potently inhibits replication of human immunodeficiency virus type 1 by interfering with the transition of the transmembrane protein, gp41, to a fusion active state following interactions of the surface glycoprotein, gp120, with CD4 and coreceptor molecules displayed on the target cell surface. Although T-20 is postulated to interact with an N-terminal heptad repeat within gp41 in a trans-dominant manner, we show here that sensitivity to T-20 is strongly influenced by coreceptor specificity. When 14 T-20-naive primary isolates were analyzed for sensitivity to T-20, the mean 50% inhibitory concentration (IC₅₀) for isolates that utilize CCR5 for entry (R5 viruses) was 0.8 log₁₀ higher than the mean IC₅₀ for CXCR4 (X4) isolates (P = 0.0055). Using NL4.3-based envelope chimeras that contain combinations of envelope sequences derived from R5 and X4 viruses, we found that determinants of coreceptor specificity contained within the gp120 V3 loop modulate this sensitivity to T-20. The IC₅₀ for all chimeric envelope viruses containing R5 V3 sequences was 0.6 to 0.8 log₁₀higher than that for viruses containing X4 V3 sequences. In addition, we confirmed that the N-terminal heptad repeat of gp41 determines the baseline sensitivity to T-20 and that the IC₅₀ for viruses containing GIV at amino acid residues 36 to 38 was 1.0 log₁₀ lower than the IC₅₀ for viruses containing a G-to-D substitution. The results of this study show that gp120-coreceptor interactions and the gp41 N-terminal heptad repeat independently contribute to sensitivity to T-20. These results have important implications for the therapeutic uses of T-20 as well as for unraveling the complex mechanisms of virus fusion and entry.