Soluble bifunctional enzyme aggregates have been prepared by cross-linking the sequential enzymes malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 4.1.3.7) using glutaraldehyde. The kinetic behaviour of this two-enzyme system in its aggregated and non-aggregated form was studied both in free solution and immobilized on Sepharose beads. This study was undertaken in order to distinguish between the following two factors which may account for the increased efficiency found in general in co-immobilized consecutive two-enzyme systems: (a) closer proximity between the participating enzymes and (b) establishment of a favourable microenvironment (such as higher local intermediate concentration caused by increased diffusional hindrance in the gel phase). It was found that in spite of a reduction of the distance between the two enzymes in the aggregated form by an estimated factor of 10(3), no kinetic advantage (shorter lag phase or higher steady-state rate) could be detected compared to the corresponding system with the two enzymes not linked to each other. However, both systems immobilized to Sepharose reached the steady-state rate of citrate formation almost immediately, in contrast to the corresponding free systems which exhibited pronounced lag phase. These results indicate that, at least in the above systems and under the conditions given, diffusional hindrance in the gel phase of the intermediate oxaloacetate, which is present in rate-limiting concentrations, is the dominant cause of the observed higher efficiency in immobilized systems.