The amplification of a substrate cycle system with reversible closed reaction of two substrates was represented by mathematical equations. The results are summarized as follows: the amplification was affected especially by the affinity of enzyme and substrate, by the rate constant in rate-limiting reaction step, and by the saturation degree of enzyme by substrate. These amplifications were not simply determined by the values of K(m) and V(max), because each rate parameter in the system can affect the degree of amplification independently. The conclusion is that the "apparent" equilibrium constant of this system cannot be uniquely estimated from only data of K(m) and V(max) even if the reaction occurs in a closed system.