To allow the detailed interpretation of the pH dependences of the steady-state parameters for the reaction catalyzed by chicken muscle triosephosphate isomerase [EC 5.3.1.1], 3 kinds of experiments were performed. First, the value of kcat/Km for enzyme-catalyzed isomerization of the phosphonate analog of D-glyceraldehyde 3-phosphate (2-hydroxy-4-phosphonobutyraldehyde) was shown to titrate with an apparent pKa of 7.5, which is close to the phosphonate''s 2nd ionization constant. Secondly, the sulfate ester analog of dihydroxyacetone phosphate (dihydroxyacetone sulfate), which exists only as a monoanion over the pH range of interest, was shown not to bind detectably to the enzyme. Thirdly, an isotopic discrimination experiment at pH 5.2 was compared with a similar investigation at pH 7.6. Both enzyme and substrate ionizations control the reaction rate in the pH range 5-8.