Pericytes and endothelial cells from the same sample of adult rat lung have been separately established in culture by use of selective growth media. The endothelial cells are positive and the pericytes negative for angiotensin-converting enzyme activity and tissue plasminogen activator. Morphologically in culture, the pericytes are similar to pericytes from bovine retina and other sites and show positive immunofluorescence to both human platelet (non-muscle) myosin and smooth muscle myosin. In this respect they resemble smooth muscle cells grown from the rat main pulmonary artery, but lack the myofilaments and dense bodies characteristic of muscle cells. Lung endothelial cells and fibroblasts are positive only for platelet myosin. Pericytes in culture demonstrate an unusual growth response to endothelial substrate, obtained by removing confluent endothelial monolayers with nonionic detergent or alkali. When plated onto this material at low density, pericyte growth is inhibited. By contrast, the substrate stimulates the growth of endothelial cells and has no effect on smooth muscle cells. Initial attachment of endothelial cells and pericytes to the substrate is similar.