In the present study, a colchicine binding assay was used to measure changes in islet polymerized and depolymerized tubulin at intervals characterizing the biphasic pattern of glucose-induced insulin release i.e., 2.5, 5.5, 10.5, and 30.5 min. At 2.5 min during the rapid onset of insulin release, a significant increase from 24% to 33% in polymerized tubulin content was observed. This increase in polymerized tubulin was followed by a reduction that temporally correlated with the disappearance of first phase release. Second phase release was also associated with a shift in equilibrium favoring tubulin polymerization at longer incubation periods. The effect of cytochalasin B on the equilibrium between polymerized and depolymerized tubulin was evaluated at a submaximal glucose concentration of 16.5 mM. Under these conditions, cytochalasin B increased polymerized tubulin content in a manner that parallels its enhancing effect on insulin release. These results indicate that the rapid events associated with the biphasic pattern of insulin release are temporally correlated with changes in polymerized tubulin.