To elucidate the mechanism of the Ca(2+)-sensitizing action of pimobendan, cardiac thin filaments were reconstituted from actin and tropomyosin-troponin complex and made to slide on a myosin layer. Although filaments showed Brownian movement with a low Ca2+ concentration, they slid at a constant velocity above a certain level of Ca2+ concentration, showing that the sliding was regulated by Ca2+ within a narrow pCa range. Acidosis, addition of inorganic phosphate, and phosphorylation of troponin I increased the threshold Ca2+ concentration. Addition of pimobendan reversed these desensitization effects. These results clearly demonstrated that pimobendan directly increases the Ca2+ sensitivity of thin filament.