Compounds that increase beta cell number can serve beta cell replacement therapies in diabetes. In vitro studies have identified several agents that can activate DNA synthesis in primary beta cells, however in small percentages of cells and without demonstration of increases in cell number. We used whole-well multi-parameter imaging to first screen a library of 1280 compounds for their ability to recruit adult rat beta cells into DNA synthesis and then assess influences of stimulatory agents on the number of living cells. The four compounds with highest beta cell recruitment were glucocorticoid-receptor ligands (GC). The GC-effect occurred in glucose-activated beta cells and was associated with increased glucose-utilization and oxidation. Hydrocortisone and methylprednisolone almost doubled the number of beta cells in two weeks. The expanded cell population provided an increased functional beta cell mass for transplantation in diabetic animals. These effects are age-dependent; they did not occur in neonatal rat beta cells where GC-exposure suppressed basal replication and was cytotoxic. It is concluded that glucocorticoids can induce replication of adult rat beta cells through a direct action, with intercellular differences in responsiveness that have been related to differences in glucose-activation and in age. These influences can explain variability in GC-induced activation of DNA synthesis in rat and human beta cells. Our study also demonstrated that beta cells can be expanded in vitro to increase the size of metabolically adequate grafts.