The pleura is comprised of a single layer of mesothelial cells resting on a complex layer of connective tissue. The ability of mesothelial cells to produce the components of this connective tissue was investigated using cultured rat mesothelial cells. These cells produced several components of extracellular matrix, including 6.8 +/- 0.2 X 10(5) collagen pro-alpha-chains per cell per hour, which represented 3.09 +/- 0.05% of all proteins synthesized by these cells. Chemical and immunologic criteria were used to demonstrate that these collagen chains included those of collagen types I, III, and IV. In addition, these cells produced elastin, as well as the connective tissue glycoproteins laminin and fibronectin. Moreover, electron microscopic studies revealed that lung mesothelial cells were capable of organizing these components into complex structures that resembled components of the extracellular matrix (thick collagen fibers, the amorphous component of elastic fibers, and basement membranelike structures), and restricted the formation of these structures to the basal region below the cells in culture. Thus, pleural mesothelial cells are active sources of a variety of connective tissue macromolecules found beneath mesothelial cells in situ, and can assemble these components into structures resembling the pleural extracellular matrix.