Pericytes are elongated cells with multiple processes and are located within the basement membrane of capillaries and postcapillary venules. Because of their shape, distribution, and morphological characteristics, pericytes are considered to be contractile cells. Cyclic GMP-dependent protein kinase (cGK) is an enzyme which mediates the effect of guanosine 3':5'-monophosphate in a second messenger cascade resulting in the phosphorylation of specific substrates. This enzyme is present in high concentration in both vascular and visceral smooth muscle as well as cerebellar Purkinje cells. Using an indirect immunoperoxidase technique, cGK was localized to both capillary and postcapillary venule pericytes in a variety of microvascular beds. Endothelial cells and connective tissue fibroblasts were consistently negative at the level of sensitivity of the procedure used. The specificity of reaction to pericytes and vascular smooth muscle within the vasculature permits cGK localization to be used as an efficient and selective staining procedure and as a criterion for identification. The presence of similar levels of cGK in both pericytes and vascular smooth muscle indicates a close biochemical relationship between these two cell types and provides further suggestive evidence that pericytes may have a contractile function similar to that of vascular smooth muscle cells.