Both the chemokine SDF-1alpha and the human immunodeficiency virus-1 (HIV-1) coat protein gp120 can bind to CXCR4 chemokine receptors but with different signaling consequences. To understand the molecular basis for these differences, we tagged the rat CXCR4 receptor with enhanced cyan (ECFP) and yellow (EYFP) derivatives of the green fluorescent protein and investigated CXCR4 receptor dimerization in human embryonic kidney (HEK)-tsA201 cells using fluorescence resonance energy transfer (FRET). Elevated FRET was detected under basal conditions from EYFP-CXCR4 and ECFP-CXCR4 receptor-transfected cells indicating a high level of CXCR4 receptor dimerization. In comparison, EYFP-CXCR4 and ECFP-mu-opioid receptor-cotransfected cells displayed a much lower FRET signal. The FRET signal resulting from EYFP-CXCR4- and ECFP-CXCR4-expressing cells could be attenuated by coexpressing nontagged CXCR4 receptors suggesting competition with fluorophore-tagged receptors in the membrane. Nontagged mu-opioid, kappa-opioid, and muscarinic receptors also decreased the FRET between the tagged CXCR4 receptor pairs but to a lesser extent. Application of the CXCR4 receptor agonist SDF-1alpha (50 nM) further increased the FRET signal from tagged CXCR4 receptors, an effect that was inhibited by the CXCR4 antagonist AMD3100. SDF-1alpha had no effect when EYFP-CXCR4 and ECFP-mu-opioid receptors were coexpressed. The effect of gp120IIIB on CXCR4 FRET was dependent on the coexpression of human CD4 (hCD4) when it increased the FRET signal, and this was decreased by AMD3100 pretreatment. FRET analysis of tagged hCD4 constructs demonstrated that there was significant association of hCD4 and CXCR4, as well as hCD4 dimerization. These data suggest that CXCR4 dimerization is involved in SDF-1alpha- and gp120-induced signaling events.