DNA replication in pancreatic beta-cells was compared in intact rats maintained hyperglycemic by continuous glucose infusion up to 10 days and in rat islets maintained in suspension culture in RPMI 1640 medium up to 12 days. Replicative activity was evaluated by counting the proportion of labeled beta-cell nuclei after injection or addition of [3H]thymidine. In both experimental systems, DNA replication initially was markedly stimulated by high glucose; then it subsided, even though high levels of glucose were maintained. Culture of pancreatic islets in suspension permitted the study of various treatments on beta-cell DNA replication. Alpha-ketoisocaproic acid, a nonglycolytic substrate for the beta-cell, stimulated DNA replication. 3-Isobutyl-1-methylxanthine (IBMX), in the presence of 4 mM glucose, was a potent stimulator. In many instances the proportion of labeled beta-cell nuclei in islets cultured with IBMX exceeded that obtained with 25 mM glucose. Dibutyryl-cyclic AMP stimulated DNA replication but was not as effective as IBMX. Although the effect of IBMX markedly decreased during the second week of culture, IBMX was much more effective than 25 mM glucose in maintaining DNA replication persistently above basal (4 mM glucose) levels.