Introduction. Mouse islets that are transplanted to the kidney lose their responsiveness to the neurotransmitters acetylcholine and noradrenaline (Shi & Ta Èljedal 1995a,b, Shi et al, 1996. In an effort to understand how islets may be affected by changes in microenvironment, we studied the insulin secretion and the muscarinic acetylcholine receptor messenger RNA (mAChR mRNA) expression of islets kept in culture for 1±7 days.Methods. Islets were isolated from BALB/c or ob/ob mice. The effects of glucose and carbachol on islets insulin secretion were studied by static incubation or dynamic perifusion methods; insulin content extracted by acid ethanol and ultrasonication; and mACh-RmRNA level were investigated by the RT-PCR method.Results. Islet insulin content was not affected by the glucose concentration in culture medium, nor by the addition of carbachol into the culture medium (average about 62 ng per lg islets dry weight). However, the insulin secretion in response to glucose was markedly decreased when islets were cultured in medium containing 2 mmol/l glucose, in comparison to islets cultured in medium containing 11.2 mmol/l (0.07 0.0 vs 2.56 0.4 ng per hr per 3 islets). The glucose-induced insulin secretions was also decreased when islets were cultured in medium containing cholinergic agaonist carbachol in comparison with islets cultured without carbachol (26.1 5.1 vs 55.9 3.5 ng per 25 min per 50 islets). Acetylcholine potentiated glucose-induced insulin secretion in fresh islets and cultured islets. This potentiating effect was diminished when islets were cultured with carbachol (5.53 + 0.7 vs 1.99 0.1 ng per 60 min per 3 islets). When measured by RT-PCR method, the expression of mAChR mRNA in pancreatic islets was up-regulated after 3-days in vitro culture. The up-regulation of mAChR mRNA was not affected by adding carbachol in culture medium, neither by culture islets in 2 mmol/l glucose.Conclusions. 1. The mAChR mRNA was up-regulated when pancreatic islets were isolated and kept in culture.The mechanism is not known, but seems to be related to the denervation states of islets, and not related to glucose concentration in the culture medium. 2. Persistent activation of mAChR by agonist carbachol decreased glucose-induced insulin secretion and diminished the potentiating efffect of acetylcholine.