Rhabdomyosarcomas have been classified on the basis of their degree of differentiation, a feature closely related with their response to chemotherapy. We recently reported the role of creatine kinase isoenzymes as tumoral markers in the diagnosis of rhabdomyosarcoma. The present study was designed to determine whether these isoenzymes are also good markers of the degree of differentiation of these neoplasm. Dimethyl sulfoxide, a well-known differentiating agent, was used to induce myogenic differentiation. This agent increased CK-MM and/or CK-MB fractions, and decreased CK-BB isoenzyme in rhabdomyosarcoma cell lines. The variable behavior of MCK-1 macromolecule was unrelated to the degree of differentiation. Our findings suggest that CK-MM, CK-MB and CK-BB isoenzymes are good markers of the degree of differentiation in rhabdomyosarcomas and could be used as specific markers of prognostic and diagnostic value.