Xylulose-, fructose-, and octulose-diphosphates are substrates for rabbit muscle aldolase with essentially identical K(m) values, but they are cleaved at different rates. After treatment with carboxypeptidase, chymotrypsin, or subtilisin, aldolase cleaves all of these substrates at the same (deceased) rate; the modified aldolase preparations are also equally impaired in their ability to catalyze the detritiation of specifically labeled dihydroxyacetone phosphate. These results suggest that aldolase exhibits "induced fit," in which the rate of cleavage is determined by the distance between the sites on the protein to which the two phosphate groups of a substrate are bound. The activity of the modified aldolases is limited by a step involving making or breaking a carbon-hydrogen bond.