Pancreatic ducts have several types of purinergic P2 receptors, however, nothing is known about P2 receptors in acini. The aim was to establish whether acini express functional P2 receptors coupled to intracellular Ca2+ signals and to measure the signals ratiometrically in a confocal laser scanning microscope. Rat acini and ducts were loaded with various Ca2+ sensing fluorophores (Fluo-4, Fura-Red, Calcium Green-1, Indo-1 and Fura-2). Only Fura-2 loaded evenly into acinar clusters and ducts and UV laser excitation at 351 and 364 nm gave signals showing opposite sensitivity to Ca2+ concentration changes. About 15% of acini or single cells within acini responded to ATP or UTP, yet all responded to carbachol. In contrast, all ducts responded to ATP/UTP. ATP stimulated uptake of a fluorescent anion, luciferin, but again only in a few acinar cells. RT-PCR analysis on acini samples collected from whole pancreas suspensions revealed transcripts for P2Y(2), P2Y(4) and P2X(1), P2X(4) receptors. The low number of functional P2 receptors in acini might be related to the fact that they release ATP. Thereby acini would avoid autocrine stimulation and initiation of autodigestive processes, such as occurs in pancreatitis.