Pancreatic stellate cells (PSCs) are the resident cells of the exocrine pancreas. In healthy pancreatic tissue they play an important role in regulation of extracellular matrix proteins turnover, and store retinoids. In response to pancreatic tissue injury PSCs undergo a phenotypic transformation and contribute to tissue fibrosis (1). Despite a growing body of evidence of the role PSCs might play in e.g. chronic pancreatitis and pancreatic cancer, relatively little is known about the exact mechanism of PSC activation and pathological transformation (2). Currently, the role of nitric oxide (NO) signaling, and its potential link with Ca2+ signaling, in these cells remains unknown and should be elucidated. In the present study NO and Ca2+ responses were examined in murine pancreatic lobules which contained both: the acinar (PACs) and the stellate cells, as well as in the cultured human PSCs line. C57BL/6 mice were humanely killed in accordance with the UK Schedule 1 of the Animals (Scientific Procedures) Act, 1986. Ca2+ signals were recorded in a ratiometric manner using Fura-2, and NO was measured with the specific fluorescent dyes: DAF-2 or DAF-FM. The simultaneous increases in intracellular Ca2+ and NO were observed in the lobular (murine) stellate cells co-loaded with DAF-2 and Fura-2 probes, after treatment with bradykinin (BK; 20 nM). Interestingly, the BK-evoked changes in the cellular levels of Ca2+ and NO showed markedly different profiles: BK caused a rapid increase in cellular Ca2+, with an initial peak and subsequent plateau, and a small, sustained increase in cellular NO. No responses were detected in the lobular PACs after BK stimulation. Hydrogen peroxide (H2O2), an oxidative pathophysiological stimulus, caused a dose dependent (0.125-0.5 mM) increase in the cellular level of NO in the lobular PSCs. A sharp increase and sustained plateau was observed after treatment with H2O2 in the higher (0.3-0.5 mM) concentration range, and a more modest increase and sustained plateau after treatment with H2O2 in the lower concentration range (0.125-0.25 mM). The development of H2O2-evoked responses was diminished substantially (p < 0.005) after the application of a NO synthase inhibitior L-NAME (0.6 mM). Other pathological stimuli, such as bile acid sodium salts: cholate (0.1-5 mM) and taurocholate (5 mM) caused elevations in cytosolic Ca2+, detected in the lobular (murine) and in the human PSCs, and related increases in cellular NO. We conclude that NO signaling, together with Ca2+ signaling, play an important role in regulation of physiological and pathological processes in the pancreatic stellate cells.