Manganese (Mn)-induced neurodegenerative toxicity has been associated with a distorted iron (Fe) metabolism at both systemic and cellular levels. In the current study, we examined whether the oxidation states of Mn produced differential effects on certain mitochondrial [Fe-S] containing enzymes in vitro. When mitochondrial aconitase, which possesses a [4Fe-4S] cluster, was incubated with either Mn(II) or Mn(III), both Mn species inhibited the activities of aconitase. However, the IC 10 (concentration to cause a 10% enzyme inhibition) for Mn(III) was ninefold lower than that for Mn(II). Following exposure of mitochondrial fractions with Mn(II) or Mn(III), there was a significant inhibition by either Mn species in activities of Complex I whose active site contains five to eight [Fe-S] clusters. The dose-time response curves reveal that Mn(III) was more effective in blocking Complex I activity than Mn(II). Northern blotting was used to examine the expression of mRNAs encoding transferrin receptor (TfR), which is regulated by cytosolic aconitase. Treatment of cultured PC12 cells with Mn(II) and Mn(III) at 100 μM for 3 days resulted in 21 and 58% increases, respectively, in the expression of TfR mRNA. Further studies on cell growth dynamics after exposure to 25-50 μM Mn in culture media demonstrated that the cell numbers were much reduced in Mn(III)-treated groups compared to Mn(II)-treated groups, suggesting that Mn(III) is more effective than Mn(II) in cell killing. In cells exposed to Mn(II) and Mn(III), mitochondrial DNA (mtDNA) was significantly decreased by 24 and 16%, respectively. In contrast, rotenone and MPP+ did not seem to alter mtDNA levels. These in vitro results suggest that Mn(III) species appears to be more cytotoxic than Mn(II) species, possibly due to higher oxidative reactivity and closer radius resemblance to Fe. Keywords manganese; iron; aconitase; Complex I; speciation; transferrin receptor; PC12 cells; mitochondria; mitochondrial DNA; cytotoxicity; Fe-S cluster Chronic manganese (Mn) intoxication in humans causes permanent neurodegenerative damage in the nigrostriatal region, resulting in a syndrome similar to Parkinson's disease (PD;Cook et al., 1974;Mena et al., 1967 Jenner et al., 1992;Sofic et al., 1991;Ye et al., 1996). A recent population study has also established that serum Fe concentrations are significantly reduced in IPD patients compared with controls, suggesting a compartment shift in Fe from blood to tissues, including brain (Logroscino et al., 1997). The role of Fe in etiopathology of IPD has been extensively reviewed by Jenner et al. (1992) and Youdim et al. (1993).Previous studies from this laboratory have established that Mn-induced neurotoxicities appear to be associated with its interaction with Fe at systemic and cellular levels (Zheng et al., 1998Zheng and Zhao, 2001). Following Mn exposure, there is a predominant influx of Fe from the blood into the cerebrospinal fluid (CSF) and from extracellular matrix to intracellular space Zheng and Zhao, 2001 et al., 1983...