The effect of the local anesthetic bupivacaine on the energy metabolism of Ehrlich ascites tumor cells has been investigated. Even at low concentrations, bupivacaine decreased the oxygen uptake rate, but its effect was remarkably higher on the uncoupled respiration. Experiments on specific segments of the respiratory chain have shown that bupivacaine did not inhibit electron transport from Q to oxygen. Spectroscopic evidences demonstrated a NAD(P)H oxidation in bupivacaine-treated cells respiring on endogenous substrates, indicating that the inhibition of oxygen depended on a reduced electron transport from site 1-entering substrates to respiratory chain. The aerobic glycolysis was stimulated by low and inhibited by high bupivacaine concentrations. The increased lactate production rate was due to an activation of mitochondrial ATPase, whereas its decrease was related to an inhibition of the hexokinase activity.