Glucose utilization and lactate production have been monitored as a function of time using 13C magnetic resonance spectroscopy and [13C1]-glucose with perfused wild type MCF-7 human breast cancer cells and a drug-resistant (AdrR) cell line derived from them. Compared to wild type cells, AdrR cells exhibited an enhanced (3-fold) rate of glycolysis, indicating an increased demand for ATP production. We have investigated the effects of glucose depletion and azide, an inhibitor of oxidative phosphorylation, on the levels of intracellular phosphates (Pi, ATP) and intracellular pH using 31P magnetic resonance spectroscopy and on the rates of glycolysis. In both cell lines, ATP levels and the rates of glucose utilization and lactate production were invariant in the presence of azide. ATP production, especially in AdrR cells, was highly dependent on active glucose metabolism. The results of these direct measurements confirm that these cells survive by predominantly utilizing glycolysis. Glutamate and myo-inositol were observed in 13C spectra of acid extracts of AdrR but not wild type cells. Both metabolites are potential substrates in drug detoxification. These differences in rates of glycolysis, ATP production, and the production of certain metabolites may reflect metabolic adaptations associated with the development of drug resistance.