Attenuation of receptor-mediated signal amplification in response to external stimuli, an essential step in the balance of cellular activation, may be mediated by receptor phosphorylation. We have recently shown that the carboxyl-terminal cytoplasmic domain of the N-formyl peptide receptor (FPR) interacts with G proteins and demonstrate here that this same region of the FPR is specifically phosphorylated by a neutrophil cytosolic kinase with properties similar to the G protein-coupled receptor kinase, GRK2. Both kinase activities show a lack of sensitivity toward protein kinase A, protein kinase C, and tyrosine kinase inhibitors but demonstrate almost identical sensitivity toward the kinase inhibitor heparin. Kinetic studies demonstrated that GRK2 has a Km for the carboxyl-terminal domain of the FPR of approximately 1.5 microM and that denaturation of the substrate results in an almost complete loss of phosphorylation. Comparative studies reveal that GRK3 has approximately 50% of the activity of GRK2 toward the FPR carboxyl terminus, whereas GRK5 and GRK6 have no detectable activity. Site-directed mutagenesis of numerous regions of the FPR carboxyl terminus demonstrated that, whereas Glu326/Asp327 and Asp333 are critical for phosphorylation, the carboxyl-terminal 10 amino acids are not required. Simultaneous substitution of Thr334, Thr336, Ser338, and Thr339 resulted in an approximately 50% reduction in phosphorylation, whereas simultaneous substitution of the upstream Ser328, Thr329, Thr331, and Ser332 or merely the Ser328 and Thr329 residues resulted in an approximately 80% reduction in phosphorylation. The introduction of negatively charged glutamate residues for Ser328 and Thr329 or Thr331 and Ser332 resulted in marked stimulation of phosphorylation. These results suggest a hierarchical mechanism in which phosphorylation of amino-terminal serine and threonine residues is required for the subsequent phosphorylation of carboxyl-terminal residues. These results provide the first direct evidence that an intracellular domain of a chemoattractant receptor is a high affinity substrate for GRK2 and further suggest a role for GRK2 or a closely related kinase in the attenuation of receptor-mediated activation of inflammatory cells.