In previous investigations we have shown a striking relationship between the activity of glycogenolytic glucose producing acid hydrolases in pancreatic islet tissue and certain insulin-releasing processes. In the present investigation we have studied the relation between islet lysosomal enzyme activities and glucose-induced insulin secretion in vitro in the presence of various insulin secretory inhibitors. It was observed that the nonmetabolizable glucose analogue, mannoheptulose (5 mmol/l) did induce a 2-fold increase in insulin release at low (1 mmol/l) glucose, and a total suppression of insulin release at high (16.7 mmol/l) glucose. These changes in the insulin-secretory pattern were accompanied by similar changes in the activity of islet acid alpha-glucosidase. The activities of neutral alpha-glucosidase (endoplasmic reticulum) or acid phosphatase and N-acetyl-beta-D-glucosaminidase (lysosomes) were not affected by mannoheptulose. 2-Deoxyglucose (5 mmol/l), another glucose analogue, did not increase insulin secretion or acid alpha-glucosidase activity at low glucose. At high glucose, however, a partial inhibition of both insulin release (approximately 50%) and acid alpha-glucosidase activity was seen. 2-Deoxyglucose slightly suppressed acid phosphatase activity but did not influence the activities of neutral alpha-glucosidase or N-acetyl-beta-D- glucosaminidase. Direct addition of glucose to islet homogenates showed a suppressive effect on alpha-glucosidase activity at pH 4.0 and 5.0. The glucose analogues displayed only marginal (-10%) inhibition of alpha-glucosidase activity at pH 5.0. No effect of the analogues was seen at pH 4.0.(ABSTRACT TRUNCATED AT 250 WORDS)