Fibroblast-like cells in the periacinar region may play an important role in periacinar fibrosis. In the present study, we isolated and cultured periacinar fibroblast-like cells (PFCs) derived from human pancreatic acini and examined the characteristics of human PFCs morphologically and immunocytochemically. Immunocytochemical study of human PFCs showed that they were positively stained with antibodies against type I collagen/procollagen, type III collagen/procollagen, fibronectin, prolyl hydroxylase beta sub-unit, type IV collagen, laminin, alpha-smooth muscle actin, vimentin, and nonmuscle myosin. Electron microscopic study showed that human PFCs contained a number of microfilaments, forming dense bodies in the cytoplasm. These results indicated that human PFCs possess characteristics of myofibroblasts. Expression of alpha-smooth muscle actin, a marker of the myofibroblast-like phenotype, was increased with time in culture and was enhanced by treatment with transforming growth factor (TGF)-beta 1. Collagen synthesis in human PFCs was stimulated by TGF-beta 1 and the proliferation of human PFCs was stimulated by platelet-derived growth factor. These findings suggest that PFCs from human pancreas seem to be involved in periacinar fibrosis.