Alnumycin A is an aromatic polyketide with a strong resemblance to related benzoisochromanequinone (BIQ) antibiotics, such as the model antibiotic actinorhodin. One intriguing difference between these metabolites is that the positions of the benzene and quinone rings are reversed in alnumycin A in comparison to the BIQ polyketides. In this paper we demonstrate that inactivation of either the monooxygenase alnT gene or the flavin reductase alnH gene results in the accumulation of a novel nonquinoid metabolite, thalnumycin A (ThA), in the culture medium. Additionally, two other previously characterized metabolites, K1115 A and 1,6-dihydroxy-8-propylanthraquinone (DHPA), were identified, which had oxidized into quinones putatively nonenzymatically at the incorrect position in the central ring. None of the compounds isolated contained correctly formed pyran rings, which suggests that on the alnumycin pathway quinone biosynthesis occurs prior to third ring cyclization. The regiochemistry of the two-component monooxygenase system AlnT/AlnH was finally confirmed in vitro by using ThA, FMN, and NADH in enzymatic synthesis, where the reaction product, thalnumycin B (ThB), was verified to contain the expected p-hydroquinone structure in the lateral ring.