We studied the human embryonal rhabdomyosarcoma cell line RD and 8 derivatives obtained in our laboratory either by cell cloning or by culturing in vitro cells from tumors or secondaries grown in nude mice. The expression of desmin and of the embryonic isoform of myosin and the formation of multinuclear myotube-like structures were studied as specific markers of myogenic differentiation. During continuous growth, each derivative contained a proportion (ranging from 5 to 80% among derivatives) of desmin-positive cells and a small number of myosin-positive or multinuclear elements. Cells from continuous cultures were injected intravenously in nude mice, producing lung and kidney/adrenal nodules. No correlation was found between the proportion of cells expressing desmin and metastatic capacity. When cultures were grown in differentiation medium (Dulbecco's minimal essential medium + 2% horse serum) some derivatives (designated type A) showed a strong increase in the proportion of myosin-positive cells, while others (type B) showed no increase. In vitro differentiation significantly reduced the metastatic ability of type A cells, while no modification was observed in type B after growth in differentiation medium. The proliferative ability of type A and type B cells grown in differentiation medium did not correlate with the proportion of myosin-positive cells, and extensive formation of multinuclear myotubes was never observed. It was concluded that reduction of experimental metastatic ability was mediated by events related to late, though not necessarily terminal, differentiation of rhabdomyosarcoma cells.