MCI-154 (6-[4-(4′-pyridylamino)phenyl]-4,5-dihydro-3(2H)pyridazinone hydrochloride trihydrate) is a potent novel cardiotonic agent whose positive inotropism is shown to be mainly based on an increase in Ca²⁺ sensitivity of the contractile apparatus. To elucidate the exact mechanism through which this drug acts, we investigated the movement of the reconstituted thin filament on a myosin layer in vitro. Cardiac thin filaments were reconstituted from actin and tropomyosin-troponin complex purified from rat cardiac acetone powder separately. Double staining of the filament showed that tropomyosin-troponin complex was integrated along actin filament homogeneously. Thin filaments thus prepared were fluorescently labeled and made to slide on rat cardiac myosin fixed on a glass coverslip while varying the [Ca²⁺] of the medium (control, pH 7.2 at 25°C). When [Ca²⁺] was low, the filaments showed only brownian motion. However, above a certain level of [Ca²⁺] (the threshold [Ca²⁺]), the filaments started to slide, and the velocity increased, reaching the maximum velocity within a very narrow range of [Ca²⁺]. The regulation was completely abolished by using simple actin filaments without tropomyosin-troponin complex, demonstrating that the regulatory proteins are responsible for this Ca²⁺ regulation of the movement of the reconstituted thin filament. Under the control condition, addition of MCI-154 shifted the threshold [Ca²⁺] to a lower level (sensitization) in a concentration-related manner. And 10⁻⁴ mol/L of MCI-154 reversed the desensitization effect induced by either acidosis (pH 6.8), low temperature (15°C), or the addition of inorganic phosphate (10 mmol/L). However, the maximum sliding velocity was not affected by the drug under any condition. In conclusion, MCI-154 directly sensitized the contractile apparatus under not only physiological but also pathophysiological conditions. This in vitro motility assay technique using reconstituted thin filaments is a useful tool for studying the mechanism of action of Ca²⁺ sensitizers.