The beta-galactosidase enzyme is a common reporter enzyme that has been used extensively in microbiological and synthetic biology research. Here, we demonstrate that caffeine and theophylline, common natural methylxanthine products found in many foods and pharmaceuticals, negatively impact both the expression and activity of beta-galactosidase in Escherichia coli. The beta-galactosidase activity in E. coli grown with increasing concentrations of caffeine and theophylline was reduced over sixfold in a dose-dependent manner. We also observed decreasing lacZ mRNA transcript levels with increasing methylxanthine concentrations in the growth media. Similarly, caffeine and theophylline inhibit the activity of the purified beta-galactosidase enzyme, with an approximately 1.7-fold increase in K-M toward o-nitrophenyl-beta-galactoside and a concomitant decrease in nu(max). The authors recommend the use of alternative reporter systems when such methylxanthines are expected to be present.