Multidrug resistance (MDR) is a common problem in the treatment of childhood rhabdomyosarcoma (RMS). A complete reversal of MDR is currently not possible. The aim of this study was to investigate the role of glutathione-S-transferase (GST) as mechanism of MDR in childhood RMS and to analyze possible reversal strategies. Female athymic mice underwent xenotransplantation with embryonal or alveolar RMS cells and were treated with vincristine. Gene expression analysis using Affymetrix HU-Gene 1.0 arrays revealed 2314 differentially expressed genes between the groups in alveolar RMS and 1387 in embryonal RMS. Ingenuity pathway analysis revealed a cluster of 5 overexpressed genes of the GST family in animals treated with vincristine, putative mediating the development of MDR. In order to analyze possible GST activity after chemotherapy with other commonly used drugs (doxorubicin, topotecan), cell culture experiments with alveolar and embryonal RMS cells were carried out. Specific GST activity was quantified using the clorodinitrobenzol conjugation with glutathione. Increased GST activity was found after incubation with cytotoxic agents in all cell lines. Highest induction of GST activity was found in embryonal RMS (up to 12-fold). After incubation with the GST inhibitors, tumor cell viability was decreased depending on the type of tumor cell and inhibitor used. We detected a novel mechanism for MDR in childhood RMS mediated via genes and proteins of the GST family. Reversal of these effects may be achieved by GST inhibitors in part. The GST family represents a promising target for further treatment strategies in childhood RMS.