Sequence alignments of mammalian microsomal (MEH) and soluble epoxide hydrolases (SEH) with bacterial haloalkane dehalogenase (HAD) and haloacetate dehalogenase (HAcD) together with structural and functional evidence suggest that these four enzymes are structurally and mechanistically related. The catalytic mechanism of HAD and MEH have been recently shown to involve an ester intermediate formed by alkylation of an active site carboxyl group. Very pronounced sequence similarities of regions of MEH, SEH, and HAcD with the active site of HAD suggest that all four enzymes belong to the same family of C-X bond hydrolases which involve an alkyl-enzyme intermediate. The catalytic triads (nucleophile-base-acid) of MEH and SEH are proposed to be Asp226-His431-Asp352 and Asp333-His523-Asp495, respectively, on the basis of sequence alignments with HAD. Although compelling arguments, through sequence alignments, can be made for the assignment of the nucleophile-base pair of the triad, the identity of the acid residue (e.g., Asp352 and Asp495) is more speculative. The three-dimensional structures of both MEH and SEH are suggested to contain structural elements of the alpha/beta hydrolase fold.