Ca²⁺-activated K⁺ channels (K_Ca) regulate vasomotor tone via smooth muscle hyperpolarization and relaxation. The relative contribution of the EDHF-mediated relaxation differs depending on vessel type and size. It is unknown whether these K_Ca channels are differentially distributed along the same vascular bed and hence have different roles in mediating the EDHF response. We therefore assessed the role of small- (SK_Ca), intermediate- (IK_Ca), and large-conductance (BK_Ca) channels in mediating acetylcholine-induced relaxations in both 1^st- and 4^th-order side branches of the rat superior mesenteric artery (MA1 and MA4, respectively). 2-mm segments of each MA were mounted in the wire-myograph, incubated with L-NAME (100 µmol/L) and indomethacin (INDO, 10 µmol/L) and contracted with phenylephrine (10 µmol/L). Cumulative concentration-response curves to ACh (0.001-10 µmol/L) were performed in the absence or presence of selective K_Ca channel antagonists. Apamin almost completely abolished these relaxations in MA4, but only partially blocked relaxations in MA1. The selective IK_Ca channel blocker TRAM-34 caused a significantly greater inhibition of the ACh-induced EDHF-mediated relaxation in MA4 compared to MA1. Iberiotoxin had no inhibitory effect in MA4, but blunted relaxation in MA1. Relative mRNA expression levels of SK_Ca (rSK1, rSK3, and rSK4 = rIK1) were significantly higher in MA4 compared to MA1. BK_Ca (rBK1 and rBK1) genes were similar in both MA1 and MA4. In conclusion, our data demonstrate regional heterogeneity in SK_Ca and IK_Ca function and gene expression, and stress the importance of these channels in smaller resistance-sized arteries, where the role of EDHF is more pronounced.