Oxidative capacity of muscles correlates with capillary density and with microcirculation, which in turn depend on various regulatory factors, including NO generated by endothelial nitric oxide synthase (eNOS). To determine the role of eNOS in patterns of regulation of energy metabolism in various muscles, we studied mitochondrial respiration in situ in saponin-permeabilized fibres as well as the energy metabolism enzyme profile in the cardiac, soleus (oxidative) and gastrocnemius (glycolytic) muscles isolated from mice lacking eNOS (eNOS^-/-). In soleus muscle, the absence of eNOS induced a marked decrease in both basal mitochondrial respiration without ADP (-32%; P<0.05) and maximal respiration in the presence of ADP (-29%; P<0.05). Furthermore, the eNOS^-/- soleus muscle showed a decrease in total creatine kinase (-29%; P<0.05), citrate synthase (-31%; P<0.01), adenylate kinase (-27%; P<0.05), glyceraldehyde-3-phosphate dehydrogenase (-43%; P<0.01) and pyruvate kinase (-26%; P<0.05) activities. The percentage of myosin heavy chains I (slow isoform) was significantly increased from 24.3±1.5% in control to 30.1±1.1% in eNOS^-/- soleus muscle (P<0.05) at the expense of a slight non-significant decrease in the three other (fast) isoforms. Besides, eNOS^-/- soleus showed a 28% loss of weight. Interestingly, we did not find differences in any parameters in cardiac and gastrocnemius muscles compared with respective controls. These results show that eNOS knockout has an important effect on muscle oxidative capacity as well on the activities of energy metabolism enzymes in oxidative (soleus) muscle. The absence of such effects in cardiac and glycolytic (gastrocnemius) muscle suggests a specific role for eNOS-produced NO in oxidative skeletal muscle.