Pericyclic reactions are powerful transformations for the construction of carbon-carbon and carbon-heteroatom bonds in organic synthesis. Their role in biosynthesis is increasingly apparent, and mechanisms by which pericyclases can catalyse reactions are of major interest(1). [4+2] cycloadditions (Diels-Alder reactions) have been widely used in organic synthesis2 for the formation of six-membered rings and are now well-established in biosynthesis(3-6). [6+4] and other 'higher-order' cycloadditions were predicted(7) in 1965, and are now increasingly common in the laboratory despite challenges arising from the generation of a highly strained ten-membered ring system(8,9). However, although enzyme-catalysed [6+4] cycloadditions have been proposed(10-12), they have not been proven to occur. Here we demonstrate a group of enzymes that catalyse a pericyclic [6+4] cycloaddition, which is a crucial step in the biosynthesis of streptoseomycin-type natural products. This type of pericyclase catalyses [6+4] and [4+2] cycloadditions through a single ambimodal transition state, which is consistent with previous proposals(11,12). The [6+4] product is transformed to a less stable [4+2] adduct via a facile Cope rearrangement, and the [4+2] adduct is converted into the natural product enzymatically. Crystal structures of three pericyclases, computational simulations of potential energies and molecular dynamics, and site-directed mutagenesis establish the mechanism of this transformation. This work shows how enzymes are able to catalyse concerted pericyclic reactions involving ambimodal transition states.