A CE-LIF detection-based assay was developed for the study of individual molecules of Escherichia coli beta-galactosidase. The assay allows for the simultaneous measurement of the electrophoretic mobility, catalytic activity and the variation in activity over time of individual enzyme molecules. In addition to showing the microheterogeneity of the enzyme molecules with respect to mobility and activity, it was demonstrated that at elevated temperatures the enzyme activity fluctuates over time. Incubation at varying temperatures showed that individual beta-galactosidase molecules exhibit differences in their change in activity upon a change in incubation temperature. Incubation at one temperature, followed by an incubation at an elevated temperature and subsequent incubation back at the initial lower temperature caused the molecules to have a different activity than they had initially. Additionally, thermal denaturation was found to cause a rapid and complete loss of activity.