Glutathione metabolism was monitored in proliferating intact, perfused MCF-7 breast cancer cells by (13)C NMR spectroscopy. Label incorporation from [3,3'-(13)C(2)]cystine in the perfusate into intracellular glutathione was monitored in native wild-type MCF-7 (MCF-7wt) cells and sublines resistant to doxorubicin (MCF-7dox) and 4-hydroperoxycyclophosphamide (MCF-7hc). Pulse-chase studies showed non-linear rates of isotope label uptake and washout. Fitting these data to an exponential model of glutathione metabolism allowed calculation of rate constants for glutathione metabolism in these cell lines. Comparison of these rate constants showed glutathione metabolism was increased in both drug-resistant lines. No significant difference was observed between these results for cells growing in three dimensions and results for cells cultured in monolayer.