A study of the substrate specificity of rabbit muscle aldolase led to the following observations. 1. 1. Both the d and l isomers of glyceraldehyde-3-phosphate can be condensed with dihydroxylacetone phospahte, but the former appears to react much more rapidly than the latter. 2. 2. Tagatose-i, 6-diphosphate, which possesses a cis configuration of hydroxyl groups on carbon atoms 3 and 4, is cleaved but at a slower rate than d-fructose-i, 6-diphosphate or l-sorbose-i, 6-diphosphate. 3. 3. The condensation of d- or l-glyceraldehyde with dihydroxyacetone phosphate to yield fructose- or sorbose-i-phosphates respectively is reversible. Crystalline rabbit muscle aldolase has a much greater affinity for fructose diphosphate (KM = 5·10−5M) than for fructose-i-phosphats (KM = 3.4 · 10−3M).