Enzyme-enzyme heteroconjugates of phosphoglyceromutase, enolase and pyruvate kinase, which sequentially act to produce ATP in the glycolytic pathway, were constructed with a bifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The conjugates of phospho-glyceromutase-enolase, enolase-pyruvate kinase and phosphoglyceromutase-enolase-pyruvate kinase were purified so as to be free from free forms of enzymes and properties of the conjugates were investigated in terms of overall reactions, and Km values and heat stability of each enzyme in the conjugates. When the initial phase of the overall reaction catalysed by a conjugate was compared with that by a mixture of the free enzymes, it was found that the lag time required for the system to achieve a steady-state was reduced in the conjugate system. This was true for all conjugates constructed. No significant increases in the Km values of the enzymes in conjugates were noted. Improvement of stability against heat was not observed for any conjugates.