Initial velocity measurements at a particular pH. and buffer concentration for enzymatic reactions involving two substrates are shown to yield an enzyme-substrate dissociation constant (k2/k1) for at least one of the substrates. A convenient method is described to determine this dissociation constant from Lineweaver-Burk plots of the data. The values calculated graphically by this method agree closely with dissociation constants determined from equilibrium binding experiments. This is true regardless of whether the dissociation constant is larger, smaller or identical with the apparent Michaelis constants. The advantages of this method are pointed out.