Recently we have shown that non-oxidative ethanol metabolites elicit a sustained rise in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i), causing pancreatic acinar cell damage (Criddle et al. 2004). We have now investigated the mechanisms by which ethanol and its metabolites influence cellular Ca²⁺ homeostasis using confocal microscopy. Experiments were carried out in freshly dispersed mouse pancreatic acinar cells (singlet, doublet or triplets) and results are expressed as mean±s.e.m. Ethanol (850 mM) produced only small, sustained increases in [Ca²⁺]_i] (115±12 nM) in the majority of cells (68%), whilst in the remainder a sharp, transient ‘spike’ increase in [Ca²⁺]_i (533±30 nM) was observed that declined to a similarly low resting level. In contrast, large and sustained (≥20 min) rises in [Ca²⁺]_i] of 546±36 nM (n=34) and 557±46 nM (n=12) were induced by the non-oxidative ethanol metabolite palmitoleic acid ethyl ester (POAEE; 100 μM; with 850 mM ethanol to maintain solubility) and palmitoleic acid (POA; 100 μM, in the absence of ethanol), respectively. The increases in [Ca²⁺]_i] induced by 850 mM ethanol, 100 μM POAEE or 50 μM POA were not inhibited by 50 μM ryanodine or 20 mM caffeine (n=6-8; p>0.05 ANOVA), indicating that neither ryanodine receptors (RyRs) nor inositol trisphosphate receptors (IP₃Rs) were involved. Under Ca²⁺-free conditions, prior release of ER Ca²⁺ stores with 2 μM thapsigargin or 10 μM acetylcholine abolished subsequent increases in [Ca²⁺]_i] by 50 μM POA and vice versa (n=17). Direct measurements of the ER Ca2+ concentration showed that both POAEE (100 μM) and POA (50 μM) emptied the ER stores (n=10), in contrast to time-control experiments in the absence of ethanol metabolites (n=16), and also markedly reduced NADH autofluorescence. POA induced complete mitochondrial depolarization, measured with tetramethyl rhodamine methyl ester (n=9). We conclude that non-oxidative ethanol metabolites increase [Ca²⁺]_i], primarily via Ca2+ release from the ER, independently of RyRs or IP₃Rs. Mitochondrial ATP depletion may be an important causal factor. These actions of the non-oxidative ethanol metabolites are most likely the principal means whereby ethanol induces acute pancreatitis.