This study examined the effects of prolactin on β-cell proliferation in pancreatic islet of Langerhans. Insulin secretion and β-cell proliferation were significantly increased from neonatal rat islets cultured for 4 days in the presence of either 500 ng/ml ovine prolactin (oPRL) or rat prolactin (rPRL). These effects could be prevented by including anti-oPRL serum in the culture media. Although insulin secretion and β-cell proliferation were slightly higher during the first 24 h of exposure to rPRL, maximal response was observed after 4 days for insulin secretion and 6-10 days for β-cell proliferation. The initial mitogenic response of β-cell to rPRL occurred by the limited recruitment of nondividing β-cells into the cell cycle and by most daughter cells proceeding directly into additional cell division cycles. Subsequently, the maximal effect of rPRL on β-cell proliferation was maintained by a higher rate of recruitment of previously nondividing β- cells into cell cycle with only one fourth of the daughter cells continuing to divide. These observations are difficult to reconcile with the proposal that a limited pool of β-cells capable of undergoing cell division exists in islets. Instead, these observations suggest that individual β-cells are transiently re-entering the cell cycle and dividing infrequently in response to rPRL. In this case, the majority of the β-cells would not be expected to be in an irreversible G(o) phase. We also demonstrated that the effects of rPRL on β-cell proliferation occur at normal serum glucose concentrations and are affected by inhibitors of polyamine metabolism. Additional studies on the effects of rPRL on β-cells should provide important information on the regulation of β-cell proliferation during conditions of increased insulin demand.