BACKGROUND & OBJECTIVE: There were a few reports about the role of PAX3-FKHR fusion gene in the oncogenesis of alveolar rhabdomyosarcoma (ARMS); however, its effect on muscle-producing factor (MPF) is not clear. This study was designed to observe the effects of fusion gene PAX3-FKHR transfection on expression of MRF4 and differentiation of cultured human rhabdomyosarcoma RD cells. METHODS: To construct an eukaryotic expression vector of PAX3-FKHR gene; the plasmids of MRF4 cDNA and PAX3-FKHR cDNA were transfected into cultured RD cells with lipofectin methods. The changes of cells growth, morphologic and the expression of MHC and alpha-actin were observed. RESULTS: There was no significant changes in RD cells after transfection with PAX3-FKHR gene in growth rate, growth manner and the expression of MHC and alpha-actin. In MRF4 expressing RD cells after transfected with PAX3-FKHR, although the expression of MRF4 was not changed, morphologic appearance showed a low differentiation; expression of MHC and alpha-actin significantly decreased. CONCLUSION: There is no obvious influence on biological character and differentiation in RD cells with transfection of PAX3-FKHR gene; PAX3-FKHR does not inhibit the expression of MRF4, but can reverse the effect of MRF4 on promoting the differentiation of RD cell.