Mitochondria isolated from rabbit soleus (98% type I) and gracilis (99% type IIb) skeletal muscle were compared for compositional differences. Whole muscle mitochondrial contents were 14.5 +/- 1.2 mg/g of wet weight in soleus and 5.3 +/- 0.6 mg/g in the gracilis muscle, a 2.7-fold difference. Maximal pyruvate plus malate oxidase activity in gracilis mitochondria was roughly 75% of that in soleus mitochondria. In contrast, glycerol 3-phosphate (G-3-P) oxidation was 10-fold greater in gracilis mitochondria. Both soleus and gracilis mitochondria exhibited additive pyruvate and G-3-P oxidase activities. In general, citric acid cycle enzyme activities were higher in soleus mitochondria. A notable exception was isocitrate dehydrogenase, which was twofold higher in gracilis mitochondria. Substrate cytochrome c reductase activities indicated that the electron transport chain (ETC) of soleus mitochondria possess roughly twice the capacity for both NADH and succinate oxidation. Similarly, the maximal activities of NADH dehydrogenase and succinate dehydrogenase were roughly twofold higher in soleus mitochondria. The findings demonstrate that mitochondria isolated from types I and IIb skeletal muscle differ substantially in composition. Furthermore, the relatively similar pyruvate plus malate oxidase activities in the face of markedly different ETC capacities suggest that the interaction between matrix dehydrogenases and the ETC may differ in mitochondria isolated from types I and IIb skeletal muscle.