Elevated serum butyrylcholinesterase (BChE) activity in the diabetic rat, mouse and human is very evident. The source of the increased level of BChE in the diabetic condition is not known. The effect of diabetes on cardiac BChE has not been studied so far, in spite of high BChE levels in the heart. In the present study, we investigated the effect of alloxan-induced diabetes on serum and on the soluble as well as the membrane-bound form of cardiac BChE activity and their substrate kinetics. We included rats of both sexes in the study. Serum BChE activity increased only in male diabetic rats (2.3-fold), while the activities of the soluble as well as the membrane-bound form of cardiac BChE activity increased 2.2- to 2.8-fold in male diabetic rats. A smaller increase (30%) was observed in the activity of the membrane-bound form of cardiac BChE in female diabetic rats. A slight reduction in BChE activity was observed in male and female rats after insulin treatment. The activity ratio of the soluble to the membrane-bound form of cardiac BChE was higher in diabetic and insulin-treated diabetic rats as compared with controls. The K(m) values of component II of the serum and soluble forms of cardiac BChE were comparable. In conclusion, the diabetes-induced increase in serum and cardiac BChE activity was sex dependent. Insulin was not able to rectify the diabetes-induced abnormalities in serum and cardiac BChE activity. The heart could be one of the possible sources of the increased level of serum BChE.