Abstract: l‐3,4‐Dihydroxyphenylalanine (l‐DOPA) is toxic for human neuroblastoma cells NB69 and its toxicity is related to several mechanisms including quinone formation and enhanced production of free radicals related to the metabolism of dopamine via monoamine oxidase type B. We studied the effect of l‐DOPA on activities of enzyme complexes in the electron transport chain (ETC) in homogenate preparations from the human neuroblastoma cell line NB69. As a preliminary step we compared the activity of ETC in cellular homogenates with that of purified mitochondria from NB69 cells and rat brain. Specific activities for complex I, complex II–III, and complex IV in NB69 cells were, respectively, 65, 96, and 32% of those in brain mitochondria. Complex I activity was inhibited in a dose‐dependent way by 1‐methyl‐4‐phenylpyridinium ion with an EC50 of ∼150 µM. Treatment with 0.25 mMl‐DOPA for 5 days reduces complex IV activity to 74% of control values but does not change either complex I or citrate synthase. Ascorbic acid (1 mM), which protects NB69 cells from l‐DOPA‐induced neurotoxicity, increases complex IV activity to 133% of the control and does not change other ETC complexes. Ascorbic acid also reverses l‐DOPA‐induced reduction of complex IV activity in NB69 cells. This observation might indicate that the protection observed with ascorbic acid is related to complex IV activation. In vitro incubation with l‐DOPA (0.125–4 mM) for 2 min produced a dose‐dependent reduction of complex IV without change in complex I and II–III activities.