Attachment and entry of HIV-1 into CD4 cells involve a series of events in which different viral envelope proteins interact with specific cell receptors, culminating in fusion of viral and cell membranes. AMD-3100 is a small molecule inhibitor of HIV-1 attachment to the CXCR4 chemokine receptor, and T-20 is a synthetic peptide corresponding to a region of HIV-1 gp41 that blocks fusion to cell membranes. To evaluate the interaction between agents acting at two different steps of the entry process, we conducted in vitro studies of the combination of T-20 and AMD-3100 against an X4 HIV-1 isolate. Single drugs or multiply diluted fixed ratio combinations of drugs were added to peripheral blood mononuclear cells infected with a clinical isolate, 14aPre. Drug interactions were evaluated using the median-effect principle and the combination index technique. The 50% inhibitory concentration (IC50) for T-20 was 0.10 &mgr;g/ml and for AMD-3100 was 0.19 &mgr;g/ml. Synergy was observed between T-20 and AMD-3100 and this increased with higher inhibitory concentrations, with combination indices ranging from 0.62 at IC50to 0.02 at IC95. Whether these synergistic interactions translate into clinical benefit will need to be addressed in the context of clinical trials.