FK506 (tacrolimus) is a secondary metabolite with a potent immunosuppressive activity, currently registered for use as immunosuppressant after organ transplantation. FK506 and FK520 are biogenetically related natural products that are synthesized by combined polyketide synthase/nonribosomal peptide synthetase systems. The entire gene cluster for biosynthesis of FK520 from Streptomyces hygroscopicus var. ascomyceticus has been cloned and sequenced. On the other hand, the FK506 gene cluster from Streptomyces sp. MA6548 (ATCC55098) was sequenced only partially, and it was reasonable to expect that additional genes would be required for the provision of substrate supply. Here we report the identification of a previously unknown region of the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 containing genes encoding the provision of unusual building blocks for FK506 biosynthesis as well as a regulatory gene. Among others, we identified a group of genes encoding biosynthesis of the extender unit that forms the allyl group at carbon 21 of FK506. Interestingly, we have identified a small independent diketide synthase system involved in the biosynthesis of the allyl group. Inactivation of one of these genes, encoding an unusual ketosynthase domain, resulted in an FK506 nonproducing strain, and the production was restored when a synthetic analog of the allylmalonyl-CoA extender unit was added to the cultivation medium. Based on our results, we propose a biosynthetic pathway for the provision of an unusual five-carbon extender unit, which is carried out by a novel diketide synthase complex.