The major regulator of the gap-1/synthesis phase (G₁/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of β-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in β-cells. Here, adenoviruses expressing cdk-4 and cyclin D₁ were used to explore rat and human pRb phosphorylation and β-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin D₁/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin D₁ caused a threefold increase in [³H]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human β-cell replication rates were increased 10-fold. Cdk-4 or cyclin D₁ overexpression did not adversely effect β-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin D₁/cdk-4 for the induction of human and rodent β-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in β-cells, β-cell lines, and stem cell-derived β-cells.