The inhibition of K⁺ channels leads to membrane depolarization and opening of voltage sensitive Ca²⁺ channels. This might mediate renal vasoconstriction and would therefore have an impact on renal blood flow (RBF). It has been demonstrated in vivo in rats that the inhibition of a single K⁺ channel does not cause a significant reduction in RBF, but that the inhibition of multiple K⁺ channels (BK_Ca, SK_Ca, K_ir, K_ATP, and K_v) causes a massive reduction in RBF. Furthermore, this reduction in RBF is abolished by the L-type Ca²⁺ channel blocker nifedipine, while this reduction is attentuated by the T-type Ca²⁺ channel blocker mibefradil. Therefore, we wished to identify if the target of these K⁺ channel and Ca²⁺ channel blockers was the afferent or efferent arteriole. To determine this, we employed the blood perfused juxtamedullary nephron preparation on mice kidneys in order to directly visualize their afferent arterioles. The simultaneous inhibition of BK_Ca, SK_Ca, K_ir, K_ATP, and K_v (by TEA, apamin, Ba²⁺, glibenclamide, and 4-aminopyridine, respectively) caused significant constriction in the afferent arteriole. We also verified the expression of these channels in mice kidneys using immunofluorescence. We conclude that it is possible to record K⁺ channel inhibitor induced afferent arteriolar vasoconstriction utilizing the blood perfused juxtamedullary nephron preparation.