The rate of glutathione synthesis was determined in drug-sensitive and -resistant MCF-7 cells by monitoring the rate of label uptake from [3,3'-13C(2)]-cystine using NMR spectroscopy and mass spectrometry. Compared with the wild-type human mammary adenocarcinoma cell line (MCF-7wt), the isotope incorporation rate was increased 1.6-, 2.4-, and 5.3-fold in the etoposide-resistant MCF-7 cell line (MCF-7vp), doxorubicin-resistant MCF-7 cell line (MCF-7adr), and 4-hydroperoxycyclophosphamide-resistant MCF-7 cell line (MCF-7hc), respectively. The increase in glutathione metabolism in the MCF-7hc line correlated with steady-state levels as determined by biochemical assay. In contrast, both the MCF-7vp and MCF-7adr lines showed increased metabolic synthesis of glutathione but displayed lower steady-state levels compared with the MCF-7wt line. The increased synthetic rates of all resistant lines reflected, in part, contributions from the increased activities of both gamma-glutamyltranspeptidase and gamma-glutamylcysteine synthetase. These results emphasize the importance of monitoring glutathione metabolic rates, rather than steady-state levels of enzymes or substrates, for assessing drug resistance.