The production and spatial organization of connective tissue components in ciliary muscle cell cultures was studied with immunohistochemical and ultrastructural methods. Antibodies against collagen types IV and VI, fibronectin and laminin were used. Laminin stains as pericellular network surrounding individual muscle cells. Type IV collagen shows positive cytoplasmic staining and only small foci of extracellular immunofluorescence. Staining for type VI collagen and fibronectin is seen near the ends of the bipolar cells, while the lateral sides of the cells remain unstained. Electronmicroscopy shows that cultured ciliary muscle cells are surrounded by an incomplete basal lamina. In addition, bundles of 5-20 nm thick extracellular microfibrils are seen. The bundles are oriented parallel to the axis of the cells and are in close contact with the cell membrane in areas where membrane-bound dense bands are formed. Immunoelectronmicroscopy indicates that the bundles contain fibronectin and type VI collagen fibrils. While the fibronectin fibrils approach the cell membrane directly, type VI collagen fibrils are usually separated from the cell membrane by fine fibrillous material of different nature. Quality and spatial organization of the extracellular material in ciliary muscle cell cultures shows marked similarities with the extracellular matrix of ciliary muscle in situ.