Neutral sphingomyelinase (nSMase)-derived ceramide has been proposed as a mediator of hypoxic pulmonary vasoconstriction (HPV), a specific response of the pulmonary circulation. Voltage-gated K⁺ (K_v) channels are modulated by numerous vasoactive factors, including hypoxia, and their inhibition has been involved in HPV. Herein, we have analyzed the effects of ceramide on K_v currents and contractility in rat pulmonary arteries (PA) and in mesenteric arteries (MA). The ceramide analog C6-ceramide inhibited K_v currents in PA smooth muscle cells (PASMC). Similar effects were obtained after the addition of bacterial sphingomyelinase (SMase), indicating a role for endogenous ceramide in K_v channel regulation. K_v current was reduced by stromatoxin and diphenylphosphine oxide-1 (DPO-1), selective inhibitors of K_v2.1 and K_v1.5 channels, respectively. The inhibitory effect of ceramide was still present in the presence of stromatoxin or DPO-1, suggesting that this sphingolipid inhibited both components of the native K_v current. Accordingly, ceramide inhibited K_v1.5 and K_v2.1 channels expressed in Ltk⁻ cells. Ceramide-induced effects were reduced in human embryonic kidney 293 cells expressing K_v1.5 channels but not the regulatory subunit K_vβ2.1. The nSMase inhibitor GW4869 reduced the thromboxane-endoperoxide receptor agonist U46619-induced, but not endothelin-1-induced pulmonary vasoconstriction that was partly restored after addition of exogenous ceramide. The PKC-ζ pseudosubstrate inhibitor (PKCζ-PI) inhibited the K_v inhibitory and contractile effects of ceramide. In MA ceramide had no effect on K_v currents and GW4869 did not affect U46619-induced contraction. The effects of SMase were also observed in human PA. These results suggest that ceramide represents a crucial signaling mediator in the pulmonary vasculature.