The glucose analogue 2-fluoro-2-deoxy-D-glucose (FDG) was used to study chemosensitivity of two human ovarian cancer cell lines and of murine L1210 cells. Cell viability was determined by measuring intracellular adenosine triphosphate (ATP) with a bioluminescence method, which has been shown to correlate closely with trypan blue, stem cell, and [3H]TdR assays. All three cell lines were sensitive to cytostatic drugs, which exerted a parallel decrease in the intracellular FDG and ATP levels. The two measures correlated positively (r = 0.66, P less than 0.001), indicating that FDG uptake is closely linked with ATP production. Relatively low hexokinase (HK)-to-glucose 6-phosphatase (HK/G6-Pase) ratios were measured, which suggests that the metabolic trapping of FDG 6-phosphate within the cytosol is incomplete. Apparently, these cell lines may not depend exclusively on glycolysis for their energy requirement. We conclude that cell killing caused by cytostatic drugs is associated with a decreased ATP content and FDG uptake. This indicates that not only ATP but also FDG may be used to study drug effects in vitro.