Abstract: AIM: To investigated the biochemical mechanisms of the positive inotropic effect of oxyphenamone(Oxy). METHODS: The assays of Na⁺,K⁺-ATPase activity, cAMP dependent phosphodiesterase(cAMP-PDE) activity and Ca²⁺-ATPase activity in sarcoplasmic reticulum(SR) isolated from cardiac muscle, cAMP level in cardiac muscle and the cardiac myofibrillar Ca²⁺,Mg²⁺-ATPase activity were adopted and compared with those of strophanthin-G（Str） and milrinone（Mil）. RESULTS: Oxy at its effective concentration, showed no remarkable inhibition on Na⁺,K⁺-ATPase and cAMP dependent phosphodiesterase (cAMP-PDE) activities, while in parallel experiments Na⁺,K⁺-ATPase and cAMP-PDE activities were significantly inhibited by Str and Mil. Their IC50 values were found to be 2.0 μmol.L^-1, and 85 μmol.L^-1, respectively. Oxy did not affect the cAMP level in cardiac muscle of guinea pig. However, Mil at 30 μmol.L^-1 in control experiments increased the cAMP level by 73.6%. These results suggest that the mechanism of the positive inotropic effect of Oxy differs from that of glycosides, PDE inhibitors and β-adrenergic agonists. Oxy at 100 μmol.L^-1 inhibited Ca²⁺-ATPase activity significantly in cardiac sarcoplasmic reticulum. Its IC₅₀ value was 200 μmol.L^-1. The result suggests that Oxy at high concentration exerts inhibitory effect on the Ca²⁺ uptake by SR. This mechanism may be partly responsible for the positive inotropic effect of Oxy. Oxy at 50 μmol.L^-1 shifted the relationship curve between pCa²⁺and myofibrillar Ca²⁺,Mg²⁺-ATPase activity to the left without affecting the maximum enzyme activity. When pCa 7, Oxy increased the myofibrillar Ca²⁺,Mg²⁺-ATPase activity in a concentration dependent manner and EC₅₀ value was about 10 μmol.L^-1. MCI-154 at 100 μmol.L^-1 and some new derivatives of Oxy with positive inotropic effect enhanced the Ca²⁺ sensitivity. Mil at 100 μmol.L^-1and some new derivatives of Oxy with no positive inotropic effect showed no effect at all. Solaro and Kitada found a positive correlation between the increase of myofibrillar Ca²⁺,Mg²⁺-ATPase activity and the enhancement of Ca²⁺ sensitivity of the contractile protein system. CONCLUSION: These results demonstrate that the biochemical mechanism of the positive inotropic effect of Oxy is different from these of the cardiac glycosides, PDE inhibitors and β-adrenergic agonists, therefore, it may be a novel cardiotonic agent, a calcium sensitizer.