Renewed interest in antifols for the treatment of childhood cancers has resulted from identification of novel antifols with broad spectrums of anti-cancer activity. We evaluated the in vitro cytotoxicity of methotrexate and pemetrexed in a panel of 12 pediatric solid tumor cell lines using the sulforhodamine-B assay. The Ewing sarcoma (ES) cell lines demonstrated the greatest sensitivity to both methotrexate and pemetrexed. Expression of folate pathway genes (DHFR, TS, GARFT, GGH) did not correlate with in vitro drug sensitivity. Further evaluation of pemetrexed for the treatment of ES is warranted. Pediatr Blood Cancer 2011; 57: 1233–1235. © 2011 Wiley Periodicals, Inc.