The response of endogenous antioxidants to acute exposure of the mitochondrial inhibitor, 3-nitropropionic acid (3-NPA), was investigated in selected rat brain regions. Rats treated with 3-NPA (30 mg/kg, s.c.) were sacrificed at 30, 60, 90 and 120 min after injection to examine the alterations in reduced glutathione levels (GSH), and activities of antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) in the hippocampus (HIP), frontal cortex (FC), and caudate nucleus (CN). CAT activity increased in the HIP 90 min after 3-NPA treatment. While cytosolic copper/zinc SOD (CuZn-SOD) and mitochondrial manganese SOD (Mn-SOD) levels increased in the FC at 120 min, only the Mn-SOD increased in the CN 90 min after treatment. The activity of GPx decreased in the HIP 120 min after 3-NPA injection. When compared with the control, administration of 3-NPA led to GSH depletion in HIP within 120 min. The depletion of GSH and induction of antioxidant enzyme activities after the 3-NPA exposure suggest conditions favorable for oxidative stress.