The role of liver mesenchymal cell populations in porcine serum-induced rat liver fibrosis were studied morphologically and immunohistochemically. Five-week-old rats were intraperitoneally injected with porcine serum twice a week and examined at various intervals between 3 and 24 wk after the initial injection. At an early phase, numbers of fibroblasts and extracellular matrix increased in the walls of central veins and in portal and capsular connective tissues. In the walls of central veins, the number of "second-layer cells" (i.e., the fibroblasts located at the second layer of the wall) increased. Connective tissue septa, accompanying some fibroblasts, extended from these interstitial tissues into the hepatic parenchyma, and their foremost edges came into direct contact with the perisinusoidal stellate cells. The sinusoids adjacent to the newly formed septa collapsed and later disappeared; this process resulted in the formation of hepatic limiting plates along the septa. At a more advanced stage, the interstitial fibroblasts and septal cells-which were derived from interstitial fibroblasts and the stellate cells-increased and became multilayered, constructing three-dimensional cell networks. These networks, together with increased collagen fibrils and elastic fibers, constitute the fibrotic dense connective tissue. In the control rat, smooth muscle cells were positive on vimentin, desmin and smooth muscle-alpha-actin staining. The stellate cells, second-layer cells, capsular and portal fibroblasts were shown to be vimentin and desmin positive and smooth muscle-alpha-actin negative. In the fibrotic liver, septal(fibroblastic) cells were vimentin and desmin positive and smooth muscle-alpha-actin negative. We conclude that not only the perisinusoidal stellate cells but also the interstitial fibroblasts, including the second-layer cells, play substantial role in the development of porcine serum-induced septal fibrosis in rat liver.