We examined whether milrinone-mediated attenuation of small mesenteric artery vasoconstriction results predominantly from the activation of vascular smooth muscle K⁺ channels. Resistance arteries (~150 µm) were dissected from rat mesentery and were mounted on a wire myograph. Isometric force development in response to increasing concentrations of norepinephrine (NE) was monitored before and after treatment with the type 3 phosphodiesterase inhibitor milrinone. Milrinone significantly reduced NE-induced vasoconstriction, attenuating both NE sensitivity and maximal tension generation. Inhibition of ATP-sensitive K⁺ channels or voltage-gated K⁺ channels did not prevent the milrinone-induced attenuation of NE responses. Blockade of inwardly rectifying K⁺ channels or Ca²⁺-sensitive K⁺ channels prevented the milrinone-mediated reduction in NE sensitivity, but this effect was apparently due to direct enhancement of vasoconstrictor responsiveness rather than interference with the mechanism of milrinone action. In addition, milrinone elicited substantial relaxation in vessels preconstricted with 100 mM KCl. This effect was mimicked by the adenylyl cyclase activator forskolin and was reversed by the Rp diastereomer of cAMP, which is a cAMP-dependent protein kinase (PKA) inhibitor. Our results indicate that cAMP/PKA-mediated impairment of vasoconstriction may occur without the contribution of K⁺ channel regulation.