In this study, we examined the inhibitory mechanism of monensin on high K+-induced contraction in guinea-pig urinary bladder. The relaxant effect of monensin (0.001-10 mu M) was more potent than those of NaCN (100 mu M-1 mM) and forskolin (3-10 mu M). Monensin (0.1 mu M), NaCN (300 mu M), or forskolin (10 mu M) inhibited high K-induced contraction without decreasing [Ca2+](i) level. Monensin and NaCN remarkably decreased creatine phosphate and ATP contents. Monensin and NaCN inhibited high K-induced increases in flavoprotein fluorescence, which is involved in mitochondrial respiration. Forskolin increased cAMP content but monensin did not. Monensin increased Na+ content at 10 mu M but not at 0.1 mu M that induced maximum relaxation. In the alpha-toxin-permeabilized muscle, forskolin significantly inhibited the Ca2+-induced contraction, but monensin did not affect it. These results suggest that the relaxation mechanism of monensin in smooth muscle of urinary bladder may be an inhibition of oxidative metabolism.