In the present study, we have investigated acetylcholine esterase (AChE) activity and muscarinic M(1), M(3) receptors kinetics in the brainstem of both young and old streptozotocin induced and insulin treated diabetic rats (D + I). Also, the functional role of acetylcholine and muscarinic receptors in insulin secretion from the pancreatic islets was studied in vitro. 90 week old control rats showed decreased V(max) (P < 0.001) for AChE compared to 7 week old control rats. V(max) was decreased (P < 0.001) in 7 week diabetic groups whereas 90 week old diabetic groups showed increased (P < 0.001) V(max) when compared to their respective controls. Binding studies using [(3)H]QNB and [(3)H]DAMP of 90 week old control showed significant increase in the B(max) (P < 0.001) and K(d) (P < 0.01) of muscarinic M(1) receptors whereas M(3) receptor number was decreased significantly (P < 0.001) with no change in affinity when compared to 7 week old control respectively. M(1) receptor number was decreased significantly (P < 0.001) whereas M(3) receptor number was increased significantly (P < 0.001) in both 7 week and 90 week old diabetic rat groups compared to their respective controls. The competition curve for [(3)H]QNB fitted for two sited model in 7 week old groups whereas fitted for one sited model in 90 week old groups. [(3)H]DAMP was fitted for two sited model in both 7 week and 90 week old groups. Insulin treatment significantly reversed (P < 0.001) the binding parameters to near control level. In vitro studies showed that acetylcholine through muscarinic M(1) and M(3) receptors stimulated insulin secretion from the pancreatic islets. Thus our studies suggest that both brainstem and pancreatic muscarinic M(1), M(3) receptors differentially regulate the cholinergic activity and insulin secretion which will have clinical significance in the management of diabetes and insulin treatment as a function of age.