Single enzyme molecule assays were performed on E. coli beta-galactosidase using a capillary electrophoresis-based protocol. Assays were performed using double incubations and two substrates, resorufin-beta-D-galactoside and DDAO-beta-D-galactoside, simultaneously. The variation between individual enzyme molecules in the ratio of product peak areas for the two different substrates used was indistinguishable from the variation in peak areas of the replicate incubations for a given enzyme molecule. This suggests that the enzyme is not heterogeneous with respect to its relative activity with the two different substrates used.