Investigation of pancreatic interstitial fibroblasts has proven difficult in situ. We have established a method for the isolation of pancreatic fibroblastoid/stellate cells by outgrowth from pancreatic tissue explanted into culture dishes. This technique gives a high yield of viable cells from small tissue samples. Outgrown fibroblastoid cells were established as a primary cell line and characterized during long-term culture. We investigated the development of stellate cell markers, i.e. fat storage, expression of desmin, and alpha-smooth muscle actin (alphaSMA), over weeks in culture. AlphaSMA, investigated by indirect immunofluorescence staining and Western blot analysis, revealed a constant rise in expression during routine culture. After 13 passages. approximately 100% of cells were positive for alphaSMA expression, indicating a myofibroblast type of differentiation in vitro.