The ability of formyl peptide receptors (FPRs) stably expressed in undifferentiated HL-60 cells to undergo ligand-induced desensitization was compared with their ability in normal and vector-transfected HL-60 cells following granulocyte differentiation with DMSO. fMet-Leu-Phe failed to induce uncoupling of FPRs from G-proteins in FPR-transfected cells, whereas uncoupling was induced in differentiated HL-60 cells and differentiated vector-transfected HL-60 cells, as determined by ligand-stimulated guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) binding and GTPgammaS inhibition of fMet-Leu-Phe binding to isolated membranes. Immunoprecipitation of Galpha(i2) from solubilized, azidoanalide (AA-gammaGTP) photolabeled membranes showed that receptors in desensitized FPR-transfected HL-60 cells remained coupled to Galpha(i2), whereas desensitized receptors in differentiated HL-60 cell membranes were uncoupled from Galpha(i2). As determined by immunoblotting, Galpha(i2) expression was similar in undifferentiated and differentiated HL-60 cells and FPR-transfected cells. Ligand-stimulated receptor internalization and desensitization of calcium redistribution were similar in all three groups of cells. Immunoblotting also indicated that G-protein-coupled receptor kinases (GRKs) 2 and 4 were present in undifferentiated FPR-transfected HL-60 cells at 50% of the level seen in differentiated HL-60 cells. However, differentiation did not increase GRK2 or GRK4 expression, indicating that differences in GRK expression do not explain deficient desensitization. The data indicated that undifferentiated HL-60 cells are unable to induce homologous desensitization of FPRs.