Emerging evidence reveals that heme oxygenase-1 (HO-1) and its product carbon monoxide (CO) can exert diverse biological and cytoprotective effects. Our group has recently identified a new class of compounds (CO-releasing molecules or CO-RMs) that can carry and deliver CO to biological systems and can be used to examine the physiological properties of CO. Here, we evaluated the influence of endogenously-generated CO (via HO-1 induction by hemin) and CO liberated from exogenously supplied CO-RMs on mitochondrial function. Renal mitochondria were isolated either from rats with increased HO-1 or from untreated animals, the latter being exposed to different concentrations of CO-RMs (10-100 microM). We found that mitochondrial oxygen uptake was significantly reduced in kidneys after HO-1 induction and, in a similar fashion, CO-RMs inhibited mitochondrial function in a concentration-dependent manner. Specifically, a marked depression of state 3 was observed resulting in a significant decrease in respiratory control index (RCI) values. When mitochondria were incubated with the inactive forms of CO-RMs, which are devoid of CO, the respiratory parameters remained unchanged. In summary, the results indicate that HO-1 induction and enhanced CO decrease renal oxygen consumption and alter mitochondrial function suggesting that CO could be a physiological regulator of mitochondrial oxidative phosphorylation.