The properties of smooth muscle cell hyperpolarization produced by acetylcholine (ACh) were investigated in mesenteric arteries isolated from mice. The resting membrane potential of the smooth muscle cells was about -60 mV. When ACh (10 microM) was applied for 1 min, the membrane hyperpolarized with a peak amplitude of about 5 mV which was reached in about 1 min, following which the potential slowly reverted to the resting level over about 7 min following withdrawal of ACh from the superfusate (recovery component). Exposure of the artery to 0.5 mM Ba(2+), an inhibitor of inward rectifier K-channels, depolarized the membrane by about 13 mV, increased the amplitude of the ACh-induced hyperpolarization to about 10 mV, and facilitated the visualization of the recovery component. Indomethacine (10 microM), an inhibitor of cyclooxygenase, inhibited the recovery component and as a consequence reduced the duration of the hyperpolarization. The ACh-induced response was not markedly altered by either N(omega)-nitro-L-arginine (10 microM), an inhibitor of nitric oxide (NO) production, or catalase (130 U/ml), a super oxide scavenger. Exogenously applied hydrogen peroxide (H(2)O(2), 300 microM) hyperpolarized the membrane by about 5 mV, which was abolished by catalase. These results suggest that in the mouse mesenteric artery, the ACh-induced hyperpolarization has two components, both an indomethacin-sensitive and an indomethacin-insensitive component. The former component may be produced by prostanoids, while the latter may be produced by factors other than NO or H(2)O(2). The results also suggested that the inward rectifier K-channels may be important for producing the resting membrane potential, but they may not be the main contributor to the ACh-induced hyperpolarization of smooth muscle cell membranes in the mouse mesenteric artery.