The effect of oxygen on the distribution of hemocyanin from Levantina hierosolima among the three sedimenting species 20, 60, and 100 S was determined under two sets of experimental conditions: (a) at pH 7.63 in the absence of Ca2+, where oxygen binding is noncooperative; (b) at pH 8.20 in the presence of 2 × 10-3 M Ca2+, where oxygen binding is cooperative. A comparison of the results in the two cases eliminates the possibility that equilibrium between species with different oxygen affinities is responsible for the cooperative behavior. Cooperative oxygen binding was demonstrated for the 20S subunits at pH 8.80 and 1 × 10-3 M Ca2+. Under these conditions, the concentration of calcium is sufficient to affect the oxygen affinity, but the concentration of calcium plus proton is not sufficient to bring about association. The findings exclude interactions among 20S subunits as a basis for cooperativity in hemocyanin.