Differentiated HL60 cells respond to challenge with ligand by mobilizing intracellular second messengers, resulting in superoxide production, degranulation, and actin polymerization with subsequent chemotaxis and phagocytosis. The functional capabilities of undifferentiated HL60 cells have not been similarly characterized due to the absence of the cell surface receptors required to initiate these processes. To investigate these properties, undifferentiated HL60 cells were transfected with one of the better characterized neutrophil chemotactic receptors, the N-formyl peptide receptor (FPR). Expression of the recombinant FPR gene product in FPR-transfected HL60 cells and the absence of the endogenous FPR in vector-transfected HL60 cells was demonstrated by Northern blot and flow cytometric analyses. FPR-transfected HL60 cells retained their ability to undergo granulocytic differentiation with dibutyryl cAMP, as determined by FMLP- and PMA-stimulated superoxide production. Furthermore, incubation of FPR-transfected HL60 cells for 5 days in the presence of FMLP resulted in limited differentiation as evidenced by the expression of functional C5a receptors. Binding studies of FPR-transfected HL60 cells demonstrated the presence of two binding affinities with dissociation constants of 0.6 and 33 nM, similar to dibutyryl cAMP differentiated HL60 cells and human neutrophils but contrasting the single high affinity state of the FPR expressed in mouse L cell fibroblasts. FPR-transfected HL60 cells displayed FMLP-dependent calcium mobilization with an EC50 of 3 nM and actin polymerization with an EC50 of approximately 10 nM. Actin polymerization was not observed in FPR-transfected L cell fibroblasts or undifferentiated vector-transfected HL60 cells. Both calcium mobilization and actin polymerization were sensitive to treatment with pertussis toxin, indicating the requirement for a Gi-like protein. Stimulation of either undifferentiated or differentiated HL60 cells with ATP resulted in pertussis toxin-insensitive calcium mobilization but was ineffective in producing actin polymerization. The results described herein show for the first time that undifferentiated HL60 cells can respond to chemoattractant receptor stimulation with many of the properties of the mature neutrophil. Transfected HL60 cells will provide an excellent system to study the characteristics of chemotactic receptors as well as the functional properties of myeloid cells.