The solute carrier protein family 12 group A, member one (Slc12a1) and two (Slc12a2) encode several splice variants of the kidney-specific and the ubiquitous isoforms, respectively, of the bumetanide (BTD)-sensitive Na-dependent K2Cl co-transporter. The Slc12a2 co-transporter is involved in the maintenance of a high intracellular chloride concentration [Cl–]i in β-cells and its inhibition with BTD blocks glucose-induced insulin secretion. In β-cells, [Cl–]i plays an important role in glucose-induced depolarization and insulin secretion. Glucose promotes electrogenic efflux of Cl– contributing to β-cell's electrical and secretory activity. To identify the expression pattern of Slc12a1 and Slc12a2 genes in β-cells we have used RT-PCR, Western blotting and immunolocalization studies in mouse pancreatic islets, β-cell lines and rat tissues. Our results demonstrate expression of specific splice variants of Slc12a1 and Slc12a2 transcripts in β-cells i.e., variants 1 of Slc12a1 (NKCC2A) and Slc12a2 (NKCC1a). Molecular cloning and characterization of Slc12a1 variant 1 transcripts from β-cells revealed an alternative splicing event involving the 5'-UTR region. NKCC2A expression at the protein level in islets and β-cells was confirmed by immunoblotting and immunolocalization. Further, NKCC2A, NKCC1a and pro-insulin co-localized in β-cells but not in the exocrine pancreas. Therefore, our results provide for the first time evidence of NKCC2A expression in pancreatic β-cells where it may play a role in insulin secretion.