Multiple impairments in HIV-1-specific cytotoxic T cells (CTL) have been reported, but derangements in HIV-1-specific CD8+ T-cell chemotaxis have not been described previously. We assessed migration to SDF-1alpha (stromal cell-derived factor 1-alpha) and CX3CL1 in vitro and expression of cognate receptors, CXCR4 and CX3CR1, by flow cytometry in peripheral blood and lymph node CD8+ T cells from HIV-1-seropositive and -seronegative individuals. Compared with seronegative individuals, percentages of CXCR4+CD8+ T cells were reduced (median, 26% versus 74%, p < 0.001) and percentages of CX3CR1+CD8+ T cells were increased (median, 33% versus 15%, p = 0.03) in seropositive individuals. Robust migration of peripheral blood mononuclear cell (PBMC) CD8+ T cells to SDF-1alpha (1 alphag/ml) was observed in both HIV-1-seropositive (median chemotactic index [CI] 4.9) and -seronegative (median CI 2.8) subjects (p = 0.46). CI to SDF-1alpha was not significantly related to percentage of CXCR4+CD8+ T cells or density of CXCR4, but correlated inversely with plasma HIV-1 RNA concentration (r = -0.82, p = 0.03). Little chemotaxis was observed in response to CX3CL1 and it was unrelated to CX3CR1 expression. Lymph node CD8+ T-cell chemotaxis to SDF-1alpha and CX3CL1 in four subjects demonstrated the same patterns observed in PBMC. HIV-1-specific tetramer-staining CD8+ T cells exhibited chemotaxis of similar magnitude as PBMC CD8+ T cells in a subset of subjects. These data suggest that SDF-1alpha is a potent chemoattractant for HIV-1-specific CTL, but that impairments in migration of HIV-1-specific CTL may exist at high viral loads. Improved understanding of the determinants of CTL localization may provide insight into novel therapies to enhance delivery of CTL to sites of HIV-1 replication.