The His-1 locus is a common site of viral insertion in murine myeloid leukemias induced by the wild mouse ecotropic retrovirus, CasBrM. In this report, we describe the cloning of a novel gene at the His-1 locus and show that His-1 expression is associated with the transformed phenotype. Northern (RNA) blot analysis identified His-1 transcripts in four transformed myeloid cell lines but in no normal tissues examined. Two of these cell lines were derived from retrovirus-induced myeloid leukemias that harbor integrated proviruses which drive His-1 gene expression by promoter insertion. The two other cell lines expressed a discrete 3-kb His-1 RNA that is derived from a novel gene consisting of three exons that span 6 kb on mouse chromosome 2. The His-1 gene is conserved as a single-copy sequence in multiple vertebrate species and is expressed as a spliced and polyadenylated RNA. A protein-coding region is not evident from analysis of the His-1 sequence because of the presence of multiple small open reading frames, none of which are greater than 219 bp. This lack of an extensive open reading frame is an unusual feature that is shared by other RNA molecules believed to function in the absence of translation.