Macrocyclic natural products are conformationally restricted molecules that often have improved ability to bind with high affinity and selectivity on a target. Within macrocycle chemistry, macrolactone formation is a particularly challenging transformation and has spurred the development of highly diverse synthetic strategies. A key strategy that is missing is a chemoenzymatic approach to this challenge, and the logical place to look for such a catalyst is the thioesterases (TEs) from the biosynthetic pathways that generate these molecules in Nature. These TEs are α/β-hydrolases containing an active site serine or cysteine and a conserved histidine/aspartate catalytic diad. The research presented here describes the development of two related TE domains from resorcylic acid lactone polyketide synthases found in various fungi. Unlike their bacterial counterparts these macrocyclization catalysts have proven to be stereotolerant with regard to the secondary alcohols involved in macrocyclization. Further work has demonstrated that they are also amenable to generating 12- to 18-member macrolactones. These TE domains can also catalyze macrolactam and cyclic depsipeptide formation, setting the stage for these enzymes to serve as a platform for catalyst development. The development of 2,3-diaminopropionate (DAP) incorporation in place of the active site Ser to trap acyl-enzyme intermediates was used to structurally characterize the formation of a macrocyclic trimer. This technique will be broadly applicable to characterizing other TEs. Overall the research presented here lays the foundation for the long term development of TEs as macrocyclization biocatalysts.