The effects of 3-acetylpyridine (3-AP) were studied in rat striatum. Striatal injections of 3-AP produced dose-dependent lesions. The lesion size was significantly increased in 4- and 12-month-old rats compared to 1-month-old rats. Coinjection of the competitive N-methyl-D-aspartate (NMDA) antagonist 2-amino-5-phosphonovaleric acid (APV) or systemic administration of the noncompetitive NMDA antagonist MK-801, the competitive NMDA antagonist LY274614, or the glutamate release inhibitor lamotrigine partially but significantly attenuated striatal lesion volume. Consistent with an NMDA receptor-mediated excitotoxic effect, histologic studies showed that 3-AP lesions result in relative sparing of NADPH-diaphorase neurons. Using freeze clamp, 3-AP resulted in a marked depletion of ATP. Two-dimensional water-suppressed proton chemical shift magnetic resonance imaging showed a striatal depletion of the neuronal marker N-acetylaspartate but no focal increase in lactate during the first 3 h after intrastriatal 3-AP injections. Pretreatment with fructose-1,6-biphosphate attenuated the lesion volume significantly, which may be due to its ability to serve as a substrate for glycolytic metabolism, with resulting ATP production. The results of the present studies support the hypothesis that 3-AP produces an impairment of energy metabolism due to its substitution for niacinamide in the formation of NAD(P). Furthermore, 3-AP toxicity may involve a secondary excitotoxic mechanism mediated by NMDA receptors.