A novel treatment for the inhibition of an enzyme-catalyzed reaction due to a ligand that combines with the substrate to form a non-productive inhibitor-substrate complex is presented. When compared to the conventional kinetic treatment of substrate depletion systems, the treatment presented here has two major advantages, namely (a) it is of general validity since no approximations are required for derivation of the pertinent equations; and (b) establishes a linear relationship between the reciprocal concentration of substrate-inhibitor complex and the ratio of free enzyme to enzyme-substrate complex concentrations, thus giving linear plots which allow the direct computation of the Km/Ki ratio. With purified enzyme preparations, this treatment allows the calculation of the absolute concentrations of all the species present in the reaction medium, although a purified preparation is not a pre-requisite for its applicability. The presented treatment has its most useful application in re-testing the Km value of an enzyme-catalyzed reaction when other experimental approaches cannot be employed.