To isolate and characterize the kinetics of variants of E. coli beta-glucuronidase (GUS) having altered substrate specificity. Two small combinatorial libraries of E. coli GUS variants were constructed and screened for improved activities towards the substrate p-nitrophenyl-beta-d-galactoside (pNP-gal). Nine of the most active variants were purified and their kinetic parameters were determined. These variants show up to 134-fold improved k(cat)/K-M value towards pNP-gal compared to wild-type GUS, up to 9 x 10(8)-fold shift in specificity from p-nitrophenyl-beta-d-glucuronide (pNP-glu) to pNP-gal compared to wild-type, and 10(3)-fold increase in specificity shift compared to a previously evolved GUS variant. The kinetic data collected for nine new GUS variants is invaluable for training computational protein design models that better predict amino acid substitutions which improve activity of enzyme variants having altered substrate specificity.