Pancreatic stellate cells (PSCs) are responsible for maintenance of the extracellular matrix in the healthy tissue as well as development of fibrosis in two major pathological conditions of the pancreas: cancer and pancreatitis. Cytosolic Ca2+ signals in PSCs have been linked to proliferation of these cells (1), but potential changes in the PSC cytosolic Ca2+ concentration, induced by agents known to be responsible for initiating acute pancreatitis, such as bile acids and products of non-oxidative ethanol and fatty acid metabolism (2), have not yet been investigated. It is well established that bile acids (BAs) and fatty acid ethyl esters (FAEEs) induce injury of pancreatic acinar cells (PACs), which is initiated by substantial liberation of Ca2+ from intracellular stores, followed by excessive Ca2+ entry (2). We have now investigated the effects of these agents on the cytosolic Ca2+ concentration in normal PSCs and compared these effects with those simultaneously measured in neighbouring acinar cells. We employed a novel approach where both PACs and PSCs are localized in freshly isolated large tissue clusters obtained after digestion of mouse pancreas with collagenase. The animals were sacrificed in accordance with Schedule 1 of the UK Animals (Scientific Procedures) Act, 1986. Confocal microscopy was applied to identify PSCs, which form three dimensional net-like structures, surrounding acini, and show a markedly stronger staining pattern with the Ca2+ sensitive dye Fluo-4 as compared with acinar cells.It has previously been shown that bradykinin induces Ca2+ signals in PSCs (1), whereas PACs are insensitive to this peptide. Therefore acute Ca2+ signal generation in response to bradykinin was used here as confirmation of recording from a PSC. The bile salt sodium cholate (1 – 5 mM) induced global and mostly sustained cytosolic Ca2+ rises in PSCs, but only modest Ca2+ oscillations in PACs. Removal of extracellular Ca2+ abolished reversibly the sustained phase of the Ca2+ elevation induced by sodium cholate in the PSCs. In contrast, taurolithocholic acid 3-sulfate (TLC-S, 200 μM) caused marked oscillatory Ca2+ responses in acinar cells, but had no or very little effect on PSCs. Both cell types responded to palmitoleic acid ethyl ester (POAEE, 200 μM) by producing substantial cytosolic Ca2+ rises, but in PSCs the responses more often took the form of global sustained elevations than in PACs.Our data indicate that PSCs and PACs respond independently, with regard to Ca2+ signalling, to pathophysiological stimuli and display very different sensitivities. Our results also shed new light on the current understanding of mechanisms promoting pancreatic pathology, which involves not only signalling in PACs but also in PSCs.