Zearalenone and radicicol are highly related resorcylic acid lactones with the rare property of having opposite stereochemical configurations of the secondary alcohol involved in lactone formation. The ability of the thioesterases from the zearalenone and radicicol biosynthetic pathways to macrocyclize both D and L configured synthetic substrate analogs was biochemically characterized and showed that both enzymes were highly stereotolerant, macrocyclizing both substrates with similar kinetic parameters. This observed stereotolerance is consistent with a proposed evolution of both natural products from a common ancestral resorcylic acid lactone.