The characteristics of Ca channels in isolated uterine smooth muscles from estradiol-treated ovariectomized rats were investigated. When the muscles were preincubated in Ca-depleted Ringer's solution for about 60 min and then immersed in Ca-depleted high K Ringer's solution or treated with acetylcholine (ACh) and serotonin (5-HT), no contractile response was observed. However, in both cases when CaCl2 was then added, a contractile response was induced. The contractile responses induced by KCl, ACh and 5-HT were inhibited dose-dependently by Ca antagonists and these inhibitions were counteracted competitively by an increase in the concentration of CaCl2. The characteristics of KCl-, ACh- and 5-HT-stimulated 45Ca uptake were investigated under the same conditions as the contractile responses. Results indicated that the contractile responses to high KCl, ACh and 5-HT induced by addition of CaCl2 in Ca-depleted Ringer's solution were dependent upon influx of Ca ions into uterine smooth muscle cells.