Initial Polytron treatment with subsequent exposure to the bacterial proteinase Nagarse has been shown to result in the isolation of two distinct populations of cardiac mitochondria, subsarcolemmal and interfibrillar mitochondria, respectively. Although these populations have been shown to possess distinct biochemical properties, few studies have been reported which document the potential differences in their response to pathological insult. We therefore examined the effect of acute hypoxia with or without reoxygenation as well as treatment with phosphate on oxidative phosphorylation on both groups of mitochondria. Freshly-isolated interfibrillar mitochondria (IFM) exhibited significantly higher respiratory values, with the exception of the ADP:O ratios, than subsarcolemmal mitochondria (SLM). With pyruvate-malate as respiratory substrate, 40 minutes hypoxia alone produced no effect on SLM whereas a stimulation in respiration was seen in IFM. A 40-minute reoxygenation period depressed the oxidative phosphorylation rate in SLM whereas it was stimulated in IFM. These treatments did not produce any effect in either population when succinate was the substrate of choice. Because of the latter observation, the possibility that increased lability of complex I of the electron transport chain accounted for the differences associated with NAD-linked substrates was studied by assessing NADH oxidation of sonicated mitochondria following the treatments. SLM exhibited enhanced permeability to exogenous NADH as well as increased sensitivity to sonication following either hypoxia or hypoxia/reoxygenation compared to IFM. Compared to hypoxia/reoxygenation, increasing concentrations of phosphate (5-15 mM) produced a marked depression in oxidative phosphorylation of SLM whereas IFM were relatively resistant. The toxic effects of phosphate were much more evident with pyruvate-malate as substrates; with succinate, oxidative phosphorylation of IFM was not depressed by phosphate whereas only a slight depression was observed with SLM. The latter population similarly exhibited reduced NADH oxidation following phosphate treatment whereas IFM were unaffected. Our studies show a differential sensitivity of two mitochondrial populations to hypoxia/reoxygenation, and, more markedly to phosphate. Since these effects were much less pronounced with succinate-linked respiration and since they were associated with defective NADH oxidation in SLM, it is suggested that the differences between the two populations may be accounted for by the increased lability of complex I of SLM due to hypoxia/reoxygenation or phosphate.