—Recent evidence suggests that functional diversity of vascular smooth muscle is produced in part by a differential expression of ion channels. The aim of the present study was to examine the role of Ca²⁺-activated K⁺ channels (K_Ca channels) in the expression of smooth muscle cell functional phenotype. We found that smooth muscle cells exhibiting a contractile function express predominantly large-conductance (≈200 pS) K_Ca (BK) channels. In contrast, proliferative smooth muscle cells express predominantly K_Ca channels exhibiting a much smaller conductance (≈32 pS). These channels are blocked by low concentrations of charybdotoxin (10 nmol/L) but, unlike BK channels, are insensitive to iberiotoxin (100 nmol/L). To determine the molecular identity of this K⁺ channel, we cloned a 1.9-kb cDNA from an immature-phenotype smooth muscle cell cDNA library. The cDNA contains an open reading frame for a 425 amino acid protein exhibiting sequence homology to other K_Ca channels, in particular with mIK1 and hIK1. Expression in oocytes gives rise to a K⁺-selective channel exhibiting intermediate-conductance (37 pS at −60 mV) and potent activation by Ca²⁺ (K_d 120 nmol/L). Thus, we have cloned and characterized the vascular smooth muscle intermediate-conductance K_Ca channel (SMIK), which is markedly upregulated in proliferating smooth muscle cells. The differential expression of these K_Ca channels in functionally distinct smooth muscle cell types suggests that K_Ca channels play a role in defining the physiological properties of vascular smooth muscle. The full text of this article is available at http://www.circresaha.org.