Protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors are effective antiretroviral drugs, but their use is associated with a high incidence of cardiovascular disease. As vascular dysfunction precedes cardiovascular events, this study aimed to examine the vascular effects of clinically used PIs (indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir) and non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine). Rat mesenteric arteries were suspended in conventional organ chambers for isometric tension recording. Efavirenz, indinavir, nelfinavir, ritonavir, and tipranavir, but not saquinavir and nevirapine, caused endothelium-independent relaxations. Lopinavir induced both endothelium-dependent and -independent relaxations; the former was inhibited by nitric oxide (NO) synthase inhibitor. Incubation with lopinavir for 24 hours reduced relaxations attributable to endothelium-derived hyperpolarization. Relaxations to the adenosine triphosphate-sensitive potassium (K(ATP)) channel opener, levcromakalim, but not those to the NO donor, sodium nitroprusside, were also inhibited. Western blotting indicated that the protein expressions of intermediate (IK(Ca)) and small (SK(Ca)) conductance calcium-activated potassium channels and K(ATP) channel were reduced in mesenteric arteries incubated with lopinavir for 24 hours. In conclusion, lopinavir differs from other PIs in that it acutely induces endothelium-derived NO-mediated relaxation. However, prolonged exposure to lopinavir impairs relaxations, likely by reducing the expressions of IK(Ca), SK(Ca), and K(ATP) channels.