The concept of endothelium derived relaxing factor (EDRF) implies that nitric oxide (NO) generated by NO synthase in the endothelium diffuses to the underlying vascular smooth muscle cells (VSMC) modulating thereby vascular tone. VSMC were regarded as passive recipients of NO from endothelial cells. However, this paradigm of a paracrine function of NO became currently subject to considerable debate. To address this issue, we examined the localization of enzymes engaged in l-arginine-NO-cGMP signaling in the rat blood vessels. Employing multiple immunocytochemical labeling complemented with signal amplification, electron microscopy, Western blotting, and RT-PCR, we found that NO synthase was differentially expressed in blood vessels depending on the blood vessel type. Moreover, the expression pattern of NO synthase in VSMC showed striking parallels with arginase and soluble guanylyl cyclase. Our findings challenge the commonly accepted view that the expression of NO synthase is restricted to vascular endothelial cells and lends further support to an alternative mechanism, by which constitutive local NOS expression in VSMC may modulate vascular functions in an endothelium-independent manner. Moreover, the co-expression of enzymes engaged in l-arginine-NO-cGMP signaling (NO synthase, arginase, and soluble guanylyl cyclase) in VSMC is indicative of an autocrine fashion of NO signaling in the vasculature in addition to the paracrine role of NO generated in the endothelium.