Three human rhabdomyosarcoma cell lines (Rh10, Rh18, and Rh28) have been established from three independently derived xenografts. These lines have been characterized as mesenchymal in origin (reactivity to desmin and vimentin antibodies) and as expressing a human fetal muscle surface antigen recognized by monoclonal antibody 5.1 H11. Measurable levels of creatine phosphokinase have been detected in the cell lines. Rh10 and Rh28 exhibit the same chromosomal translocation and express an atypical lactate dehydrogenase isoenzyme which may be homologous to those previously reported in other tumor types. The karyotype analysis has confirmed that each cell line was derived from its respective tumor and thus provides a unique model for future investigations.