Immunohistochemistry shows that skeletal muscle fibers express endothelial-type (ec)-NOS in a heterogeneous pattern distinct from that of neuronal (nc)-NOS. Analysis of adjacent serial sections reveals: 1) a strong correlation of ec-NOS expression to mitochondrial content (visualized histochemically by succinate dehydrogenase); 2) lack of correlation of ec-NOS to fiber ATPase type (in contrast to nc-NOS); and 3) constitutive co-expression of ec- and nc-NOS in some fibers. Preparations of mitochondria from diaphragm exhibited calcium-dependent NOS activity, which functioned to inhibit in vitro oxygen consumption. The results establish that normal skeletal muscle cells can express two constitutive isoforms of NOS (nc- and ec-) and suggest a functional role for ec-NOS in oxidative, mitochondria-rich fibers.