Recent studies have shown that two different voltage-dependent Ca²⁺ channels are expressed in pancreatic islets, the β-cell/neuroendocrine-brain and the cardiac subtypes. The effects of chronic hyperglycemia on the levels in pancreatic islets of the mRNAs encoding the α₁-subunits of the β-cell and cardiac subtype Ca²⁺ channels were studied in rats made hyperglycemic by infusion of glucose for 48 h. A competitive reverse transcriptase-polymerase chain reaction procedure was used to obtain quantitative data on the levels of these two transcripts in islets obtained from individual rats. The quantitative polymerase chain reaction data indicate that the levels of mRNA encoding the α 1-subunit of the β-cell Ca²⁺ channel are 2.5-fold > those for the cardiac subtype. The levels of β-cell Ca²⁺ channel mRNA were 72.9% lower in the glucose-infused animals when compared with the saline-infused animals (P < 0.005) and those of the cardiac channel were 72.1% lower in the animals infused with glucose (P < 0.02). In contrast, glucose infusion resulted in a twofold increase in insulin mRNA levels and did not significantly alter levels of β-actin mRNA. In situ hybridization studies revealed that the mRNAs for these two Ca²⁺ channels are expressed at higher levels in normal rat islets than in the surrounding acinar tissue, which suggests that the observed changes in mRNA levels occur within cells of the pancreatic islet. To assess the possible functional consequences of this reduction in expression of mRNA for the Ca²⁺ channels, the insulin secretory responses of perfused pancreases to the Ca²⁺ channel agonist Bay K8644 were studied. In pancreases from glucose-infused animals, the relative incremental insulin secretory response to Bay K8644 was reduced, and a similar blunting of the acute response to glucose also was seen. In summary, the predominant voltage-dependent Ca²⁺ channels subtype expressed in islets under basal conditions and after glucose infusion is the β-cell form with lower levels of the cardiac subtype. Glucose infusion for 48 h results in a significant reduction in the mRNA levels for both the β-cell and cardiac subtypes, and this is associated with enhanced basal secretion but reduced responses to glucose and the Ca²⁺ channel agonist Bay K8644.