A possible role for the transfer of reducing equivalents between mitochondrial and cytoplasmic pools in the process of nutrient-stimulated insulin secretion was investigated by exposing rat pancreatic islets to aminooxyacetate, which inhibits transamination reactions and, hence, could impair the operation of the malate-aspartate shuttle. Aminooxyacetate (5.0 mM) decreased by 72-79% the transamination of exogenous pyruvate, L-leucine, or 2-ketoisocaproate but failed to affect the oxidation of glucose, L-leucine, or 2-ketoisocaproate and reduced by no more than 28-31% the oxidation of L-glutamine. In the 0.5-5.0 mM range, aminooxyacetate caused a dose-related inhibition of nutrient-stimulated insulin release, and lowered the cellular malate to pyruvate ratio. In the absence of both extracellular Ca2+ and exogenous nutrient, aminooxyacetate also inhibited the insulin release evoked by the combination of Ba2+ and theophylline. These findings suggest that a transfer of reducing equivalents to extramitochondrial sites participates in the process of insulin release, whether the latter involves the oxidation of exogenous or endogenous nutrients.