Macrolide antibiotics such as azithromycin and erythromycin are mainstays of modern antibacterial chemotherapy, and like all antibiotics, they are vulnerable to resistance. One mechanism of macrolide resistance is via drug inactivation: enzymatic hydrolysis of the macrolactone ring catalyzed by erythromycin esterases, EreA and EreB. A genomic enzymology approach was taken to gain insight into the catalytic mechanisms and origins of Ere enzymes. Our analysis reveals that erythromycin esterases comprise a separate group in the hydrolase superfamily, which includes homologues of uncharacterized function found on the chromosome of Bacillus cereus, Bcr135 and Bcr136, whose three-dimensional structures have been determined. Biochemical characterization of Bcr136 confirms that it is an esterase that is, however, unable to inactivate macrolides. Using steady-state kinetics, homology-based structure modeling, site-directed mutagenesis, solvent isotope effect studies, pH, and inhibitor profiling performed in various combinations for EreA, EreB, and Bcr136 enzymes, we identified the active site and gained insight into some catalytic features of this novel enzyme superfamily. We rule out the possibility of a Ser/Thr nucleophile and show that one histidine, H46 (EreB numbering), is essential for catalytic function. This residue is proposed to serve as a general base in activation of a water molecule as the reaction nucleophile. Furthermore, we show that EreA, EreB, and Bcr136 are distinct, with only EreA inhibited by chelating agents and hypothesized to contain a noncatalytic metal. Detailed characterization of these esterases allows for a direct comparison of the resistance determinants, EreA and EreB, with their prototype, Bcr136, and for the discussion of their potential connections.