With subfragment-one of myosin used as the histochemical marker, actin filament distribution was mapped in cells of the aqueous outflow pathway. In uveal, corneal-scleral and juxtacanalicular meshwork cells, bundles of actin filaments were present along the basal cytoplasm. Some of these bundles terminated at adhesion plaques. Juxtacanalicular meshwork cells contained, in addition to bundles, randomly oriented actin filaments at the end of cell extensions. Giant vacuoles in the inner wall of Schlemm's canal did not have actin filaments associated with their membranes; there were, however, intermediate filaments present along the membrane. Bundles of actin filaments were also found within the cells of both inner and external walls of Schlemm's canal. Cells of both the meshwork and inner wall of Schlemm's canal had actin filaments extending out into the cytoplasm from cell junctions. In all areas of the meshwork and especially in the area external to Schlemm's canal, cells were observed which had a cytoplasm, very rich in actin filaments. These cells had characteristics common to smooth muscle cells. The actin filaments of cells of the aqueous outflow pathway could have any or all of several functions. Bundles of filaments in the meshwork could contract to lift and separate trabecular sheets and/or, through their association with adhesion plaques, they could stabilize cells on their collagen substrate, thus acting as cytoskeletal struts. Randomly oriented actin filaments, noted particularly in cells of the juxtacanalicular meshwork, may play a role in phagocytosis. Actin filaments found in association with junctions may be important structures for maintaining cell-to-cell contacts. Lack of actin filaments around giant vacuoles in Schlemm's canal indicates that they do not play a role in shuttling aqueous across the endothelium of the canal. The significance of "actin-rich" cells in the meshwork and those external to Schlemm's canal is unknown.