The T-type Ca2+ channel is an important determinant of electrical activity and of Ca2+ influx in rat and human pancreatic β-cells. We have identified and sequenced a cDNA encoding a T-type Ca2+ channel α1-subunit derived from INS-l, the rat insulin-secreting cell line. The sequence of the cDNA indicates a protein composed of 2,288 amino acids that shares 96.3% identity to α1G, the neuronal T-type Ca2+ channel subunit. The transmembrane domains of the protein are highly conserved, but the isoform contains three distinct regions and 10 single amino acid substitutions in other regions. Sequencing rat genomic DNA revealed that the α1-subunit we cloned is an alternative splice isoform of α1G. By using specific primers and reverse transcription-polymerase chain reaction, we demonstrated that both splice variants are expressed in rat islets. The isoform deduced from INS-1 was also expressed in brain, neonatal heart, and kidney. Functional expression of this α1G isoform in Xenopus oocytes generated low voltage- activated Ba2+ currents. These results provide the molecular biological basis for studies of function of T-type Ca2+ channels in β-cells, which is where these channels may play critical roles in diabetes.