ATP and other nucleotides are released from various cells, but the pathway and physiological stimulus for ATP release are often unclear. The focus of our studies is the understanding of ATP release and signaling in rat exocrine pancreas. In acinar suspension mechanical stimulation, hypotonic shock and, most importantly, cholinergic stimulation released 5-20nM ATP into the medium, as monitored by luminescence of the luciferin/luciferase reaction. ATP release was visualized at the single acinus level as luciferin consumption detected by confocal laser scanning microscopy (CLSM). The estimated ATP concentrations were higher, about 10µM, around acinar cells after cholinergic stimulation. Fluorescence of quinacrine and MANT-ATP indicated that some ATP is stored in secretory granules. Pancreatic acini have transcripts for P2X₁, P2X₄, P2Y₂, and P2Y₄ receptors, but measurements of Ca²⁺ signals in isolated acini using Fura-2 and CLSM revealed that only about 15% of acini respond to extracellular ATP or UTP. Hence, in acini only a few P2 receptors are functional and the distribution seems heterogenous. In contrast, pancreatic ducts have transcripts for P2Y₂, P2Y₄, P2X₄, and P2X₇ receptors that consistently increase intracellular Ca²⁺. Patch-clamp studies show that P2Y₂/P2Y₄ receptors inhibit K⁺ channels, and thus downregulate secretion. P2X₄/P2X₇ channels stimulate Na⁺/Ca²⁺ influx and may upregulate secretion by yet unknown mechanisms. Taken together, our studies show that pancreatic acini release ATP, primarily in response to cholinergic stimulation, and thus ATP may be a paracrine mediator between pancreatic acini and ducts. Drug Dev. Res. 59:128-135, 2003. © 2003 Wiley-Liss, Inc.