Immune-deprived female CBA/CaJ mice bearing xenografts of six different human rhabdomyosarcoma lines were treated with 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU). Tumor responses were compared with levels of O6-methylguanine-DNA methyltransferase activity because of evidence indicating that repair of DNA interstrand cross-link precursors, mediated by the transferase, may be an important determinant of MeCCNU cytotoxicity. Levels of methyltransferase in tumor extracts were measured by determining the loss of O6-methylguanine from 3H-labeled methylated DNA. Five of the six tumor lines examined showed either no response to MeCCNU or regrowth after an incomplete response. In each instance, the extent of tumor regression correlated with the level of O6-methylguanine-DNA methyltransferase activity in tumor extracts. The single highly drug sensitive line was totally devoid of the activity. These results suggest that O6-methylguanine-DNA methyltransferase levels in human tumor cells may be a clinically useful predictor of sensitivity to the chloroethylnitrosoureas.