A number of hypotheses on the etiology of Parkinson's disease and other CNS disorders postulate a role of metal ions and/or neuromelanin. As part of an investigation of the interactions between neuromelanin and metal ions, we have studied the amount and type of metal ions in human neuromelanin in intact substantia nigra and in isolated neuromelanin using electron paramagnetic resonance (EPR), which selectively measures metal ions which are in valence states that have unpaired electrons and total reflection X-ray fluorescence (TXRF), which measures total metals. EPR also is a principal technique for studying the biophysics of melanins by analysis of its free radicals. The studies of substantia nigra with TXRF indicated the presence of substantial amounts of iron, zinc, lead, copper, manganese, and titanium at concentrations up to 4 times greater than those of non-pigmented brain tissue (basis pedunculi). The concentrations of metal ions in isolated neuromelanin were 5-260 times higher than in substantia nigra. The studies with EPR indicated that there were substantial amounts of paramagnetic metals ions, especially iron, bound to neuromelanin in intact substantia nigra, and the presence of these metal ions modified the EPR spectra of the free radicals of neuromelanin. We conclude: 1. Compared to other regions of the mid-brain, the substantia nigra contains increased amounts of many different metal ions; 2. Many of these metal ions are in paramagnetic valence states; 3. There are high concentrations of paramagnetic metal ions bound to neuromelanin. These results are consistent with the hypotheses that postulate a role of metal ions in promoting oxidative reactions in pigmented neurons.