31P-Magnetic resonance spectroscopy has been used to assess the changes in the levels of water-soluble phosphate pools in T47-D breast carcinoma cells induced by the antimitochondrial drugs, gossypol and 6-aminonicotinamide. A decrease in the NTP/Pi ratio occurred after treatment with gossypol. No change in the NTP/Pi ratio occurred on treatment with 6-aminonicotinamide; however, a substantial accumulation of 6-phosphogluconate was observed. Pretreatment of T47-D cells with gossypol prevented the accumulation of 6-phosphogluconate. This facile and non-invasive approach suggests that the oxidative part of the pentose-phosphate shuttle is an important source of reducing equivalents in T47-D cells. This pathway may prove to be a useful target for site-directed drug attack in carcinoma cell lines that require large quantities of NADP for the synthesis of fatty acids and steroids.