Rationale HIV-1 co-receptors, CXCR4 and CCR5, are essential for HIV entry and its life cycle. Expression of CXCR4 on T-lymphocytes is lower during HIV-infection and is normalized by antiretroviral therapy. We propose that HIV-1 Nef, an important HIV-1 regulatory protein, may play an important role in HIV-1 co-receptor expression on T lymphocytes during HIV infection. Methods CXCR4 and CCR5 expression on CD4 + T-lymphocytes was measured using quantitative flow cytometric assay in 23 HIV-infected children and 10 healthy subjects after 24-hour in vitro stimulation with various HIV-1 regulatory proteins (Nef 100 ng/ml, Tat 1 ng/ml, Gag 500 ng/ml, and gp120IIIB 250 ng/ml). Results Without HIV-1 regulatory protein stimulation, mean CXCR4 expression on CD4 + T-lymphocytes was significantly higher in healthy subjects compared to HIV-infected children (36.79% vs 24.60%; p=0.005). Following 24-hour incubation with Nef, mean CXCR4 expression on T-lymphocytes of healthy subjects was significantly decreased when compared to the expression on cells without stimulation (30.38% vs. 36.79%; p=0.012). There was no longer significant difference in mean CXCR4 expression on T-lymphocyte between HIV-infected children and healthy subjects after Nef stimulation (24.81% vs. 30.4%; p=NS). There was no significant change in mean CXCR4 expression on T-lymphocytes after stimulation with other HIV-1 proteins in HIV-infected children or healthy subjects. No significant change in the CCR5 expression on CD4 + T-lymphocytes was seen after stimulation with various HIV-1 proteins. Conclusions HIV-1 Nef protein decreases CXCR4 expression on CD4 + T-lymphocytes in healthy subjects. This may be an important mechanism for CXCR4 down-regulation during HIV-1 infection.