The binding of small peptide ligands to high affinity chemoattractant receptors on the surface of neutrophils and monocytes leads to activation of heterotrimeric G-proteins, stimulation of phosphatidylinositol-phospholipase C (PI-PLC), and subsequently to the inflammatory response. It was recently shown (Amatruda, T. T., Gerard, N. P., Gerard, C., and Simon, M. I. (1993) J. Biol. Chem. 268, 10139-10144) that the receptor for the chemoattractant peptide C5a specifically interacts with G alpha 16, a G-protein alpha subunit of the Gq class, to trigger ligand-dependent stimulation of PI-PLC in transfected cells. In order to further characterize this chemoattractant peptide signal transduction pathway, we transfected cDNAs encoding the formylmethionylleucylphenylalanine receptor (fMLPR) into COS cells and measured the production of inositol phosphates. Ligand-dependent activation of PI-PLC was seen in COS cells transfected with the fMLPR and G alpha 16 and stimulated with fMLP but not in cells transfected with receptor alone or with receptor plus G alpha q. Chimeric receptors in which the N-terminal extracellular domain, the second intracellular domain, or the intracellular C-terminal tail of the fMLP receptor was replaced with C5a receptor domains (Perez, H. D., Holmes, R., Vilander, L. R., Adams, R. R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295) were capable of ligand-dependent activation of PI-PLC when co-transfected with G alpha 16. A chimeric receptor exchanging the first intracellular domain of the fMLPR was constitutively activated, stimulating PI-PLC in the absence of ligand. Constitutive activation of PI-PLC, to a level 233% of that seen in cells transfected with wild-type fMLP receptors, was dependent on G alpha 16. Site-directed mutagenesis of the first intracellular domain of the fMLPR (amino acids 54-62) reveals this to be a domain necessary for ligand-dependent activation of G alpha 16. These results suggest that different receptors which mediate similar biochemical responses may utilize distinct mechanisms to activate G-proteins. Differences among the signaling pathways triggered by chemoattractant factor receptors suggest an opportunity for pharmacologic modifications of the inflammatory response.