Genome sequencing has revealed that fungi have the ability to synthesize many more natural products (NPs) than are currently known, but methods for obtaining suitable expression of NPs have been inadequate. We have developed a successful strategy that bypasses normal regulatory mechanisms. By efficient gene targeting, we have replaced, en masse, the promoters of nonreducing polyketide synthase (NR-PKS) genes, key genes in NP biosynthetic pathways, and other genes necessary for NR-PKS product formation or release. This has allowed us to determine the products of eight NR-PKSs of Aspergillus nidulans, including seven novel compounds, as well as the NR-PKS genes required for the synthesis of the toxins alternariol (8) and cichorine (19).