Voltage-gated potassium channels (Kv channels) are involved in repolarization of excitable cells. In pancreatic β-cells, prolongation of the action potential by block of delayed rectifier potassium channels would be expected to increase intracellular free calcium and to promote insulin release in a glucose-dependent manner. However, the specific Kv channel subtypes responsible for repolarization in β-cells, most importantly in humans, are not completely resolved. In this study, we have investigated the expression of 26 subtypes from Kv subfamilies in human islet mRNA. The results of the RT-PCR analysis were extended by in situ hybridization and/or immunohistochemical analysis on sections from human or Rhesus pancreas. Cell-specific markers were used to show that Kv2.1, Kv3.2, Kv6.2, and Kv9.3 are expressed in β-cells, that Kv3.1 and Kv6.1 are expressed in α-cells, and that Kv2.2 is expressed in δ-cells. This study suggests that more than one Kv channel subtype might contribute to the β-cell delayed rectifier current and that this current could be formed by heterotetramers of active and silent subunits.