Single enzymemolecule assays on E. coli beta-galactosidase were performed using a capillary electrophoresis-based method. Three types of assays were performed. The catalytic rate of 20 individual molecules was assayed in duplicate in the presence of 50 mu M substrate. The ratio of rates for the second incubation relative to the first was 0.96 +/- 0.03, showing the reproducibility of the method. In the second assay, the rates were determined in the absence and presence of 210 mu M L-ribose, a competitive inhibitor. The ratio of the rate in the presence of inhibitor to that in its absence for 19 individual molecules was 0.44 +/- 0.23. This large relative standard deviation suggests that each individual enzyme molecule was affected to a different extent by the presence of the inhibitor, which is consistent with K-I being heterogeneous. To estimate K-I for individual molecules, a third assay was performed. Each molecule was incubated in the presence of 30 and 50 mu M substrate and then in the presence of 50 mu M substrate plus 210 mu M inhibitor. Comparison of the rates in the two substrate concentrations allowed for the determination of the individual Km of eachmolecule. From this value and the difference in rates in the presence and absence of inhibitor, the individual molecule K-I values were determined. This value was found to differ between individual molecules and was found to increase with an increase in Km. Modeling showed that a heterogeneity in K-I results in an alteration in the Michaelis-Menten curve for a population of enzymes in the presence of a competitive inhibitor.