Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerolphosphocholine; PAF) induces leukocyte accumulation and activation at sites of inflammation via the activation of a specific cell surface receptor (PAFR). PAFR couples to both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins to activate leukocytes. To define the role (s) of G i and G q in PAF-induced leukocyte responses, two G-protein-linked receptors were generated by fusing Gα i3 (PAFR-Gα i3) or Gα q (PAFR-Gα q) at the C terminus of PAFR. Rat basophilic leukemia cell line (RBL-2H3) stably expressing wild-type PAFR, PAFR-Gα i3, or PAFR-Gα q was generated and characterized. All receptor variants bound PAF with similar affinities to mediate G-protein activation, intracellular Ca 2+ mobilization, phosphoinositide (PI) hydrolysis, and secretion of β-hexosaminidase. PAFR-Gα i3 and PAFR-Gα q mediated greater GTPase activity in isolated membranes than PAFR but lower PI hydrolysis and secretion in whole cells. PAFR and PAFR-Gα i3, but not PAFR-Gα q, mediated chemotaxis to PAF. All three receptors underwent phosphorylation and desensitization upon exposure to PAF but only PAFR translocated βarrestin to the cell membrane and internalized. In RBL-2H3 cells coexpressing the PAFRs along with CXCR1, IL-8 (CXCL8) cross-desensitized Ca 2+ mobilization to PAF by all the receptors but only PAFR-Gα i3 activation cross-inhibited the response of CXCR1 to CXCL8. Altogether, the data indicate that G i exclusively mediates chemotactic and cross-regulatory signals of the PAFR, but both G i and G q activate PI hydrolysis and exocytosis by this receptor. Because chemotaxis and cross-desensitization are exclusively mediated by G i, the data suggest that differential activation of both G i and G q by PAFR likely mediate specific as well as redundant signaling pathways.