We have isolated a murine cDNA by virtue of its expression in pre-B cell lines but not in plasmacytomas. In mouse, mRNA is detected by Northern blot analysis exclusively in hematopoietic tissues with the exception of brain. When expressed in bacteria, the encoded protein is specifically recognized by several antibodies against the murine hematopoietic differentiation marker called heat-stable antigen (HSA). In contrast to previous speculations, these results suggest that the antigenic determinant recognized by anti-HSA antibodies is proteinaceous. The cDNA sequence is essentially identical to a mouse HSA cDNA recently published (Kay et al., J. Immunol. 1990. 145: 1952). Three HSA genes were isolated, one of which corresponds to the isolated cDNA. The other two appear to be intronless retroposons which, despite numerous sequence changes, maintain an open reading frame. One of these intronless genes could code for an alternative form of HSA with a transmembrane domain and a cytoplasmic tail not encoded by the intron-containing gene. This gene could be expressed, based on sequence comparisons of the three genes. However, using polymerase chain reaction amplification of first strand cDNA from various cell lines and adult mouse tissues, we could detect expression only of the intron-containing gene.