Using a monoclonal antibody to the interleukin 3 (IL-3) receptor (anti-Aic2), we isolated a cDNA (AIC2B) from a mouse mast cell line which is homologous to the previously characterized gene for the IL-3 receptor (AIC2A). This cDNA encodes a polypeptide of 896 amino acid residues and has 91% amino acid sequence identity with the IL-3 receptor. A consensus sequence defining an additional cytokine receptor family is present in this clone. Compared to the AIC2A clone, the AIC2B cDNA encodes a protein with amino acid substitutions, insertions, and deletions dispersed throughout the entire protein. Oligonucleotide probes specific for each cDNA hybridized with different genomic fragments, indicating that the AIC2A and AIC2B proteins are encoded by two distinct genes. Fibroblasts transfected with the AIC2B cDNA expressed the protein at the cell surface as determined by binding with the anti-Aic2 antibody but did not bind IL-3 or other cytokines, including IL-2, IL-4, granulocyte-macrophage colony-stimulating factor, erythropoietin, and IL-9 (p40) at concentrations between 1 and 10 nM. An S1 nuclease protection assay was used to discriminate between the AIC2A and AIC2B transcripts. We found that the AIC2B gene was coexpressed with the AIC2A gene. These results suggest a potential involvement of AIC2B in cytokine signal transduction.