We have investigated the effects of glucose and the polypeptide growth factor growth hormone (GH), platelet-derived growth factor (PDGF), insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and transforming growth factor alpha (TGF alpha) on the polyamine content, in relation to proliferation and insulin secretion and content, of pancreatic beta-cells. Fetal rat pancreatic islets containing a high proportion of beta-cells were cultured for 3 days with growth factors. beta-cell replication was significantly increased by glucose, GH, and PDGF plus IGF-I in parallel with increased islet polyamine contents. In contrast, neither EGF nor TGF alpha influenced the islet DNA synthesis rate, polyamine content, insulin content, or insulin accumulation in culture medium. When the increased polyamine content evoked by growth-promoting agents was prevented by inhibitors of polyamine synthesis, elevated DNA synthesis rates persisted or were even augmented. However, subcellular fractionation analysis of islet homogenates revealed that the nuclear polyamine content was not affected by the inhibitors. On the other hand, islet insulin content and glucose-regulated insulin release were decreased by polyamine synthesis inhibitors. Glucose oxidation rates remained unchanged, suggesting that inhibitors were not toxic to islet cells. We conclude that prevention of increases in total cellular content of polyamines in response to glucose, GH, or PDGF plus IGF-I does not prevent mitogenicity of these growth factors. However, when their synthesis is inhibited normal levels of polyamines seem to be maintained in the cell nucleus, an event that may be sufficient to permit a mitotic signal to be translated into a proliferative response.