Synthesis-secretion coupling of insulin was determined in perfused pancreases stimulated for 3 h by various sugars. These monosaccharide stimuli included glucose alone at either 200 or 300 mg/dl; mannose or fructose alone at 1,200 mg/dl; or combinations of mannose and fructose or galactose and fructose at 600 mg/dl each. Glucose and mannose each promoted insulin synthesis and secretion. Mannose at 1,200 mg/dl produced synthesis-secretion coupling similar to glucose at 200 mg/dl. Fructose alone at 1,200 mg/dl failed to cause any significant release of insulin, but it did significantly increase beta cell insulin content. When mannose and fructose were combined at 600 mg/dl each, in the absence of glucose, they resulted in a synergistic effect on insulin secretion and an additive effect on insulinogenesis, which was in excess of, or equal to, the insulinotropic effect of glucose at 300 mg/dl. These results clearly establish that the synthesis and secretion of insulin can be uncoupled. Mannose primarily stimulates the putative beta cell glucoreceptor, and fructose signals the insulin biosynthetic pathway. When combined, these monosaccharides couple synthesis-secretion of insulin comparable to glucose. The data suggest that the uncoupling of insulin secretion and synthesis, which may contribute either independently or in combination to abnormalities in pancreatic function observed in various diabetic conditions can be studied using the isolated perfused pancreas model. Use of this relatively physiological experimental model should provide optimal opportunity to further investigate and identify cellular controlling signals regulating either insulin biosynthesis, insulin secretion, or the coupling of both mechanisms.