Neuronal nitric oxide synthase (NOS I) is a Ca2+/calmodulin–binding enzyme that generates nitric oxide (NO•) and L-citrulline from the oxidation of L-arginine, and superoxide (O2•−) from the one-electron reduction of oxygen (O2). Nitric oxide in particular has been implicated in many physiological processes, including vasodilator tone, hypertension, and the development and properties of neuronal function. Unlike Ca2+, which is tightly regulated in the cell, many other divalent cations are unfettered and can compete for the four Ca2+ binding sites on calmodulin. The results presented in this article survey the effects of various divalent metal ions on NOS I–mediated catalysis. As in the case of Ca2+, we demonstrate that Ni2+, Ba2+, and Mn2+ can activate NOS I to metabolize L-arginine to L-citrulline and NO•, and afford O2•− in the absence of L-arginine. In contrast, Cd2+ did not activate NOS I to produce either NO• or O2•−, and the combination of Ca2+ and either Cd2+, Ni2+, or Mn2+ inhibited enzyme activity. These interactions may initiate cellular toxicity by negatively affecting NOS I activity through production of NO•, O2•− and products derived from these free radicals.