The anatomic distribution of smooth muscle myosin, a contractile protein, was determined in a variety of lymphoid tissues (spleen, lymph nodes, tonsils) with the use of highly specific rabbit antibodies to human uterine smooth muscle myosin and an indirect immunoperoxidase technique. In the spleen, in addition to the anticipated immunoreactivity in the walls of arteries, veins, splenic capsule, and trabeculas, other staining patterns were observed. Smooth muscle myosin-containing cells which comprised the adventitia of the trabecular arteries appeared continuous with myosin-containing reticular cells of the white pulp. The latter cells assumed a circumferential pattern within the periarteriolar lymphoid sheaths, then blended delicately with the red pulp at the marginal zone. Ultrastructurally, immunogold techniques demonstrated that smooth muscle myosin in these cells was localized to cytoplasmic filaments. Within the red pulp, a different and distinct staining pattern was observed for the splenic sinuses. Short, regular, orderly, and repetitive bands of immunoreactivity, aligned parallel to the long axis of the sinus, extended between contiguous ring fibers. By immunoelectron microscopy these structures corresponded to distinct bundles of filaments in the endothelial lining cells of the splenic sinuses. Factor VIII associated antigen was also identified in the splenic lining cells in cryostat and paraffin sections, and ultrastructurally. Within the red pulp of the spleen, the sheaths of sheathed capillaries also revealed strong immunoreactivity for smooth muscle myosin. Other sites of immunohistochemical localization of smooth muscle myosin included dendritic reticulum cells present in reactive follicles and in nodular non-Hodgkin's lymphomas. Certain vascular structures, specifically sinus lining cells and Schweigger-Seidel capillary sheaths of the spleen and postcapillary venules of lymph nodes and tonsils, coexpressed smooth muscle myosin and Factor VIII associated antigen. The patterns of localization of smooth muscle myosin are correlated with anatomic structures and possible tissue functions.