Hematopoietic progenitor cells of the A/J strain of mice show a pronounced defect in the ability to form colonies or proliferate in response to interleukin-3 (IL-3). Comparison of immunoblots of A/J mast cells and of mast cells from the C57BL/6 strain that respond normally to IL-3 showed that, in both strains, a 125-kD band of the expected size was recognized by an antibody against the beta chain of the IL-3 receptor, the AIC2A molecule. However, in the C57BL/6 cells, there was an additional 110-kD species not seen in cells of the A/J strain. Analyses using bone marrow-derived mast cells from a panel of A/J x C57BL/6 and A/J x C57BL/6 recombinant inbred (RI) mice showed that the hypo-responsiveness to IL-3 is governed by a single gene. However, the absence of this 110-kD species in the A/J strain did not co-map with IL- 3 hypo-responsiveness but did indeed map to the AIC2A genetic locus. These data show that this trait in the A/J strain was due to a polymorphism of the AIC2A gene unrelated to IL-3 hypo-responsiveness. Typing of the RI strains for the markers D14Mit98, D14Mitl4, and D14Mit133 mapped the locus determining hypo-responsiveness to IL-3 to the subtelomeric region of chromosome 14, the region that also bears the gene encoding the alpha chain of the IL-3 receptor (lL-3Ralpha). Immunofluorescence analyses indicated that IL-3Ralpha protein was undetectable on fresh bone marrow cells from A/J mice, although clearly detectable on cells from the responder C57BL/6 strain. However, IL- 3Ralpha was readily detectable at normal levels on A/J mast cells generated by culture of A/J bone marrow cells in a combination of IL-3 and steel factor. Moreover, IL-3Ralpha on these A/J mast cells appears to be functional in that IL-3 stimulation of these cells results in tyrosine phosphorylation events characteristic of IL-3 signaling, including tyrosine phosphorylation of the beta chain of the IL-3 receptor, Jak-2 kinase, and SHPTP2. Collectively, these data indicate that the hypo-responsiveness of A/J mice to IL-3 is due to a defect in the gene encoding IL-3Ralpha and that, although this defect gives rise to reduced expression of alpha chain on primary bone marrow cells, this defect is not absolute and that, under certain circumstances, A/J cells can express functional receptors.