Glucose-stimulated insulin secretion is associated with transients of intracellular Ca²⁺ concentration [Ca²⁺]_i in the pancreatic β-cell. We identified the expression and function of specific small-conductance Ca²⁺-activated K⁺ (SK) channel genes in insulin-secreting cells. The presence of mRNA for SK1, -2, -3, and -4 (intermediate-conductance Ca²⁺-activated K⁺ 1 [IK1]) channels was demonstrated by RT-PCR in rodent islets and insulinoma cells. SK2 and -3 proteins in mouse islets were detected by immunoblot and immunocytochemistry. In the tTA-SK3 tet-off mouse, a normal amount of SK3 protein was present in islets, but it became undetectable after exposure to doxycycline (DOX), which inhibits the transcription of the tTA-SK3 gene. The SK/IK channel-blockers apamin, dequalinium, and charybdotoxin caused increases in average [Ca²⁺]_i levels and in frequency of [Ca²⁺]_i oscillations in wild-type mouse islets. In SK3-tTA tet-off mice, the addition of apamin with glucose and tetraethylammonium (TEA) caused a similar elevation in [Ca²⁺]_i, which was greatly diminished after DOX suppression of SK3 expression. We conclude that SK1, -2, -3, and IK1 (SK4) are expressed in islet cells and insulin-secreting cells and are able to influence glucose-induced calcium responses, thereby regulating insulin secretion.