Although several attempts have been made to establish a relationship between glucose concentration and pentosephosphate shunt (PPS) activity of pancreatic islets in vitro, no such relationship has been found. Recently it has been shown that exogenous insulin, when added in vitro, inhibited islet PPS activity. Since it was possible that the failure to detect such a relationship was due to insulin released into the incubation medium, we reinvestigated the subject, introducing GPAIS (guinea pig antiinsulin serum) to bind insulin in the medium. When five islets were incubated with 16.7 mM glucose for 90 min, the absolute rate as well as the percentage of islet PPS activity were found to be significantly higher (P less than 0.05) in the presence of GPAIS than in its absence. In the presence of GPAIS, the absolute rate and the percentage of islet PPS activity increased in a dose-related manner (18, 43, and 124 pmol/five islets.90 min and 2.3%, 3.2%, 9.4%, respectively) when the glucose concentration was raised from 5.6 to 11.1 and 16.7 mM. Our data, therefore, indicate that the activity of islet PPS activity in vitro depends on the concentration of glucose, provided that insulin does not accumulate in the incubation medium. This suggests that PPS activity might play a role in glucose-induced insulin secretion. However, whether the PPS contributes to the secretory mechanism by producing a signal for insulin secretion or by forming cofactors required by the secretory mechanism remains to be established.