Enzyme inhibitors have played an important part in advancing our knowledge of biochemistry. This is especially true for those inhibitors which function as substrate analogues and have been used to elucidate metabolic pathways and kinetic mechanisms of enzyme-catalysed reactions. Most studies, with reversible inhibitory substrate analogues have been performed under steady-state conditions where the concentration of enzyme is very much less than that of the inhibitor (and substrate) and where all the equilibria involving the enzyme and reactants are set up rapidly. However, there are inhibitors that do not satisfy one or either of these two conditions. As far back as 1943, it was recognized that there are compounds which inhibit enzyme-catalysed reactions at concentrations comparable to that of the enzyme and under conditions where the equilibria are set up rapidly 1. Such compounds are referred to as tight-binding inhibitors. For the derivation of rate equations that describe tight-binding inhibition, allowance must be made for the reduction in the inhibitor concentration (It) that occurs on formation of an enzyme-inhibitor (El) complex. No longer does the fh'st steady-state assumption hold. The concentration of EI is not negligibly small compared with It and the free concentration of inhibitor is not equal to its added concentration. The resulting equation, which is a quadratic function with squared and linear terms in velocity, pre-