A subtractive cloning procedure was used to characterize the molecular changes involved in transformation of normal myoblasts to rhabdomyosarcoma (RMS) cells. Here we describe the cloning of DRAL, a novel LIM-domain protein expressed in primary myoblasts but down-regulated in the RMS cell line RD. DRAL is a LIM-only protein with five LIM domains whereby one LIM domain consists only of the second half of the consensus motif. Interestingly, down-regulation of DRAL was not confined to the RD RMS cells, but was a phenomenon extended to other RMS cell lines of both embryonal and alveolar subtype, and to some breast cancer cell lines. Analysis of the expression pattern in normal human tissues revealed that DRAL is expressed at high levels in the heart, suggesting an important function in the specification of the terminally differentiated phenotype of heart muscle cells. Immunofluorescence studies using an antibody directed against recombinant DRAL localized the protein predominantly in the nucleus of cultured cells. On the basis of these results, we conclude that down-regulation of DRAL correlates with the tumor phenotype of RMS cells.