The relationship between cellular metabolism and signalling is an important problem in physiology. Acetylcholine as one of the main secretagogues modulates mitochondrial functions in acinar pancreacytes (Voronina et al., 2002; 2004; 2010), presumably due to increase of ATP hydrolysis or Ca2+-transport into mitochondria. The aim of this work was to investigate mechanisms of acetylcholine analogue, carbamoylcholine (CCh), action on respiration and oxidative phosphorylation of isolated pancreatic acini. Male rats (200-300 g) were anaesthetised with chloroform, decapitated and following the removal of the pancreas suspensions of isolated acini (cell viability ≥ 95%) were obtained (Williams et al., 1978). Oxygen consumption was measured using a Clark electrode at 37°C in a standard extracellular solution (140.0 mM NaCl, 4.7 mM KCl, 1.3 mM CaCl2, 1.0 mM MgCl2, 10.0 mM HEPES, 10.0 mM glucose, 2.5 mg ml-1 BSA and 0.1 mg ml-1 soybean trypsin inhibitor at pH 7.4). Respiration of digitonin-permeabilized cells was measured in sucrose-based solution (Manko et al., 2012). Statistical significance was determined by paired t-test. Values are given as mean±S.E.M. Resting respiration rate of isolated acini was 0.26±0.02 nmol O2 min-1 10-6 cells. CCh (10 μM), added into respiration chamber (rapid but transient effect), intensified respiration by 23.4 % for 33±3 s (P≤0.001, n=15). This effect was completely prevented by preceding application of 10 μM 2-aminoethoxydiphenyl borate (2-APB), 6 μM oligomycin or 0.5 μM FCCP (n=3-4). Ruthenium red (10 μM) attenuated rapid CCh effect (~5%, P≤0.01, n=3), however it was prolonged to 74±10 s. Incubation (5 min) with 1 or 10 μM CCh (slow long-term effect) elevated respiration rate by 11.4 or 12.4%, respectively (n=3). 0.1 μM CCh did not influence respiration rate. Preincubation (10 min) with 2-APB or ruthenium red did not affect resting respiration rate and prevented slow CCh effects (n=3-4). Oligomycin inhibited respiration rate by 62.7% (P≤0.001, n=4), and abolished slow effects of CCh. FCCP increased respiration rate by 48.12% in control (P≤0.001, n=4). After preincubation with 1 μM CCh, FCCP-uncoupled respiration rate increased by 8.75%. Also, preincubation with CCh prior to cell permeabilization increased respiration rate at pyruvate+malate oxidation by 10.5% (P≤0.05, n=5), but not at succinate oxidation. In contrast, preincubation with 10 μM CCh did not influence FCCP-uncoupled respiration and decreased respiration at pyruvate+malate oxidation by 12% (P≤0.05, n=5). Thus, mediocre CCh dose (1 μM) intensifies respiration and oxidative phosphorylation of acinar pancreacytes by feedforward mechanism via Ca2+-transport into the mitochondria and activation of Ca2+-sensitive mitochondrial dehydrogenases. Prolonged action of high CCh dose (10 μM) seems to impair mitochondrial functions.