A critical problem in the storage and delivery of pharmaceutical proteins is aggregation in the solid state induced by elevated temperature and moisture. These conditions are particularly relevant for studies of protein stability during accelerated storage or for proteins loaded in polymeric delivery devices in vivo. In the present investigation, we have found that, when exposed to an environment simulating these conditions, lyophilized insulin undergoes both covalent and noncovalent aggregation. The covalent process has been elucidated to be intermolecular thiol-catalyzed disulfide interchange following beta-elimination of an intact disulfide bridge in the insulin molecule. This process is accelerated by increasing the temperature and water content of the insulin powder or by performing lyophilization and/or dissolution of insulin in alkaline media. The aggregation can be ameliorated by the presence of Cu2+, which presumably catalyzes the oxidization of free thiols. The water sorption isotherm for insulin reveals that the extent of aggregation directly correlates with the water uptake by the lyophilized insulin powder, thus pointing to the critical role of protein conformational mobility in the aggregation process.