Culture of isolated rat islets at either 5.5 or 11 mM glucose for up to 6 days was associated with significant time-dependent increases in glyceraldehyde 3-phosphate dehydrogenase (G3PD) activity of islet homogenates compared with freshly isolated islet G3PD activity. In addition, after 6 days of culture of islets at 11 mM glucose, there was a significant increase in G3PD activity compared with the enzyme activity of islets cultured at 5.5 mM glucose. Culture of islets at 5.5 mM glucose for 2 days in the presence of forskolin, 3-isobutylmethylxanthine (IBMX), and 8-bromo-cyclic AMP also significantly increased G3PD activity compared with control islets, although there was no change in enzyme activity after only 1 day of culture with forskolin. Treatment with forskolin was associated with an increase in the Vmax of G3PD, but no change was observed in the apparent K(m) with NAD. IBMX and 8-bromo-cyclic AMP also increased G3PD activity in islets cultured at 11 mM glucose for 2 days. 8-Bromo-cyclic AMP did not affect or inhibit G3PD activity when added directly to islet homogenates. Islets cultured with 8-bromo-cyclic GMP for 2 days at 5.5 or 11 mM glucose did not show changes in G3PD activity. Increases in G3PD activity did not correlate with significant changes in islet glucose utilization. Thus, G3PD activity is modulated by the duration of glucose stimulation in cultured islets, and cyclic AMP may mediate changes in G3PD activity in islet cells.