The equivalence of the four dihydroxyacetone phosphate binding sites of aldolase was abolished by lowering the temperature. At pH 6.2 and -13 degrees C, four binding sites were detected by gel filtration; two sites with a Kdiss less than or equal to 0.1 microM, and a second set of sites with a Kdiss = 4 microM. The alteration of the binding was accompanied by the alteration of the catalytic activity. The low-affinity sites were incapable of catalyzing the cleavage of the (3S) C-H bond of dihydroxyacetone phosphate, and form only the ketimine phosphate intermediate. The high-affinity sites were still able to cleave the (3S) C-H bond of dihydroxyacetone phosphate; however, the eneamine phosphate intermediate formed was almost fully converted into the eneamine-aldehyde . . . phosphate intermediate, which was the prevailing species at the equilibrium. The mechanism of the half-of-the sites reactivity of aldolase at low temperature has been explained and the nonequivalence of sites in promoting catalysis has been utilized to dissect and characterize the individual partial reactions of the enzyme. In the course of these studies it has been shown that the rate of hydration-dehydration of dihydroxyacetone phosphate at -24 degrees C was too slow to measure.