The effect of O-2 concentration on mitochondrial nitric oxide synthase (mtNOS) activity and on O-2 production was determined in rat liver, brain, and kidney submitochondrial membranes. The K-mO2 for mtNOS were 40, 73, and 37 muM O-2 and the V-max were 0. 5 1, 0.49, and 0.42nmol NO/min mg protein for liver, brain, and kidney mitochondria, respectively. The rates of O-2(-) production, 0.5-12.8 nmol O-2(-)/min mg protein, depended on O-2 concentration up to 1.1 mM O-2. Intramitochondrial NO, O-2(-), and ONOO- steady-state concentrations were calculated for the physiological level of 20 muM O-2; they were 20-39 nM NO, 0.17-0.33 pM O-2(-) , and 0.6-2.2nM ONOO- for the three organs. These levels establish O-2/NO ratios of 513-1000 that correspond to physiological inhibitions of cytochrome oxidase by intramitochondrial NO of 16-25%. The production of NO by mtNOS appears as a regulatory process that modulates mitochondrial oxygen uptake and cellular energy production. (C) 2003 Elsevier Science (USA). All rights reserved.