Anti-HIV therapeutic agents are divided into reverse transcriptase inhibitors, protease inhibitors (PI), integrase inhibitors, and entry inhibitors (CCR5 inhibitors). The combination therapy of HIV drugs with different mechanisms referred to as suppressing the proliferation of HIV in the long term. Unfortunately, HIV PI are accompanied by side effects in long-time treatment. The side effects are HIV PI-induced insulin-resistance, diarrhea and nausea. However, the effects of HIV PI on muscle contraction on gastrointestinal smooth muscle is still unknown. In the present study, we examined the effects of HIV PI on muscle contraction in the rat and guinea pig ileum. 1) In rat and guinea pig ileum, indinavir (3-100 μM) or ritonavir (0.1-10μM) inhibited high K⁺- or histamine-induced contraction, dose-dependently, but the inhibition in high K⁺-induced contraction more strongly than that in histamine-induced contraction. 2) In guinea pig ileum, ritonavir inhibited high K⁺-induced increases of intracellular Ca²⁺ level and contraction. 3) In guinea pig ileum, ritonavir inhibited high K⁺- or histamine-induced contraction of PSS including pyruvate instead of glucose. However, ritonavir -induced relaxation did not differ that of PSS including glucose. These results suggested that HIV PI-induced relaxation in ileum probably due to the inhibiting Ca²⁺ influx.