Rat pancreatic monolayer cultures were utilized to study the effects of glucose, tolbutamide, dexamethasone phosphate, bovine growth hormone and porcine glucagon on beta cell replication. Cultures were established in medium 199 (glucose concentration 1 mg. per milliliter) containing 10 per cent fetal bovine serum. They were subsequently incubated for two consecutive two day intervals in this same control medium or in media containing the test agents. Cultures were then labeled with 3-H-thymidine and reincubated in control medium for two days to permit beta cell regranulation, thus facilitating staining. Replication was estimated by determining the frequency of labeling of aldehydethionin positive (AT+) cells in stained radioautographs. Both glucose (3 mg. per milliliter medium) and tolbutamide (100 μ. per milliliter) caused a three- to fourfoldincrease in labeling compared to controls (14 labeled AT+ cells per 500 AT+ cells). In -contrast, dexamethasone (10 μ. per milliliter) produced a two-thirds decrease in labeling. Growth hormone (10 μ. per milliliter) and glucagon (10 μ per milliliter) produced no statistically significant effects. Alterations in labeling correlated with changes in the quantity of insulin released into the medium during each of the two day incubation intervals. Both glucose and tolbutamide increased insulin release; dexamethasone decreased insulin release, while growth hormone and glucagon produced no significant effects. These data suggest that agents which stimulate insulin release can trigger beta cell replication in vitro. In addition, since growth hormone, glucocorticoid and glucagon stimulate beta cell replication in vivo but not in vitro, this action in vivo may be mediated by effects on other tissues rather than by direct action on the beta cell.