Our aim was to prepare an optimum formulation for a sustained-release preparation of insulin using biodegradable polymer composed of co-poly(D,L-lactic/glycolic) acids (PLGA)(L/G ratio: 50/50). Various kinds of PLGA microcapsules containing 3% insulin were administered subcutaneously (250 U/kg) as a single dose to rats with streptozotocin-induced diabetes, and plasma insulin levels were monitored. The following results were obtained. (1) Glycerin and water were suitable additives to prepare a reproducible injectable formulation. (2) Addition of zinc compounds was essential to diminish rapid insulin release and six-fold molar excess of ZnO to insulin was desirable. (3) The size of insulin particles showed the following order: human insulin > lyophilized human insulin > Zn-free human insulin. Zn-free insulin was similar to lyophilized insulin with respect to control of rapid release, so a smaller particle size was essential. (4) The size of the microcapsules also affected the release of insulin. With larger microcapsules (approximately 30 microm), there was gradual release and a significant second phase of insulin release, while smaller microcapsules did not allow sustained release. Some variation in microcapsule size contributed to more constant and sustained release. (5) Based on the insulin release profile in vivo, a suitable molecular weight for PLGA was around 6000. The biological activity of insulin extracted from the formulation was similar to that of normal insulin. These experiments allowed us to prepare a desirable sustained-release insulin formulation.