Rat pancreatic periacinar fibroblastoid cells (PFCs) appear to be involved in intralobular fibrosis and acinar cell regeneration. We isolated pancreatic acini of the rat, cultured the fibroblastoid cells, and characterized the cells morphologically and immunohistochemically. Isolated acini were seeded on culture dishes, and spindle-shaped cells migrated and proliferated. On Electronmicroscopic examination, microfilament bundles were seen, and the intracellular localization of vimentin, alpha-smooth muscle actin, and non-muscle myosin was identified immunohistochemically. These findings strongly suggest that the cells were myofibroblast-like. The PFCs were also demonstrated, immunohistochemically, to contain prolyl hydroxylase, type-I procollagen, type-III procollagen, type-IV collagen, fibronectin, and laminin. Stimulation by transforming growth factor beta 1 (TGF beta 1) increased intracellular immunoreactive prolyl hydroxylase and collagen synthesis in the PFCs. These findings indicate that PFCs proliferate in culture as myofibroblast-like cells and synthesize extracellular matrix components. It is possible that PFCs are involved in intralobular fibrosis in response to stimulation with TGF beta 1.