G-protein-coupled receptors (GPCR) are prime targets for therapies with small molecule-antagonists. Since yeast have GPCR triggered signaling pathways analogous to those present in mammalian cells, it is possible to express human receptors in yeast coupled to the pheromone responsive signaling cascade in variants that contain mammalian-yeast Galpha subunit chimeras. CXCR4 and CXCR4(N119S), a constitutively active mutant were expressed in yeast coupled to pheromone responsive reporter genes, HIS3, lacZ, or FUI, and tested for signaling activity. Compounds derived from T140, an inverse agonist for CXCR4, were screened for activity using yeast cells expressing CXCR4(N119S) and containing a FUS1-lacZ reporter gene. Levels of inhibition of beta-galactosidase activities triggered by constitutive activation of the pheromone response pathway that were obtained in the presence of the T140 derived compounds correlated with affinities measured in radioligand binding inhibition experiments. The yeast signaling system may provide an effective approach for screening chemokine receptor antagonists.