The course of the enigmatic iterative use of a polyketide synthase module was deduced from targeted domain inactivation in the aureothin assembly line. Mutational analyses revealed that the N-terminus of AurA is not involved in the iteration process, ruling out an ACP-ACP shuttle. Furthermore, an AurA(KS°, ACP°)-AurA(AT(0)) heterodimer proved to be nonfunctional, whereas aureothin production was restored in a ΔaurA mutant complemented with AurA(KS°)-AurA(ACP°). This finding supports a model according to which the ACP-bound polyketide intermediate is transferred back to the KS domain on the opposite PKS strand.