The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation. Since previous studies indicated that glucose-stimulated activation of mammalian target of rapamycin (mTOR) leads to [3H]thymidine incorporation and that mTOR activation is mediated, in part, through the KATP channel and changes in cytosolic Ca2+, we determined whether glyburide, an inhibitor of KATP channels that stimulates Ca2+ influx, modulates [3H]thymidine incorporation. Glyburide (10-100 nM) at basal glucose stimulated [3H]thymidine incorporation to the same magnitude as elevated glucose and further enhanced the ability of elevated glucose to increase [3H]thymidine incorporation. Diazoxide (250 μM), an activator of KATP channels, paradoxically potentiated glucose-stimulated [3H]thymidine incorporation 2-4-fold above elevated glucose alone. Cell cycle analysis demonstrated that chronic exposure of islets to basal glucose resulted in a typical cell cycle progression pattern that is consistent with a low level of proliferation. In contrast, chronic exposure to elevated glucose or glyburide resulted in progression from G 0/G1 to an accumulation in S phase and a reduction in G2/M phase. Rapamycin (100 nM) resulted in an ∼62% reduction of S phase accumulation. The enhanced [3H]thymidine incorporation with chronic elevated glucose or glyburide therefore appears to be associated with S phase accumulation. Since diazoxide significantly enhanced [3H] thymidine incorporation without altering S phase accumulation under chronic elevated glucose, this increase in DNA synthesis also appears to be primarily related to an arrest in S phase and not cell proliferation.