Myosin-I is a nonfilamentous motor protein associated with the actin cytoskeleton and cellular membranes in several cell types. The occurrence and subcellular distribution of myosin-I in mammalian and fish RPE were investigated to examine the possible role of myosin-I in retinal pigment epithelium (RPE) motility processes.Antibodies directed against myosin-I proteins from bovine adrenal medulla or chicken intestinal brush border were used to examine cultured fetal human RPE cells and freshly isolated bovine or green sunfish RPE by Western immunoblots and immunocytochemistry, using both conventional and confocal fluorescence microscopy.The monoclonal antibody directed against bovine adrenal myosin-I identified a single strong immunoreactive band at 116 kD in Western blots of homogenates of cultured human RPE cells and 114 kD in bovine RPE sheets. An immunoreactive band of similar molecular weight was also observed in bovine and rabbit retina. Cell fractionation studies of bovine RPE cells revealed that myosin-I was present in all fractions that included cell membranes. The polyclonal antibody directed against chicken brush border myosin-I identified doublet immunoreactive bands at 115/110 kD in Western blots of homogenates of fish retina but identified a strong predominant immunoreactive band at 140 kD in fish RPE and brain homogenates; minor bands at 115/110 kD were identified in fish RPE homogenates. Immunocytochemistry of cultured human RPE cells using the bovine adrenal myosin-I antibody revealed a broad distribution of myosin-I that appeared to be most concentrated along the length of the lateral membranes; no colocalization was seen with actin-rich stress fibers.Proteins immunoreactive with myosin-I antibodies are present in both RPE and retina of mammals and green sunfish. In confluent cultures of human RPE cells, myosin-I is concentrated along the lateral cell membranes of the cuboidal cells.