Myxobacteria show a high potential for the production of natural compounds that exhibit a wide variety of antibiotic, antifungal, and cytotoxic activities. The genus Sorangium is of special biotechnological interest because it produces almost half of the secondary metabolites isolated from these microorganisms. We describe a transposon-mutagenesis approach to identifying the disorazol biosynthetic gene cluster in Sorangium cellulosum So ce12, a producer of multiple natural products. In addition to the highly effective disorazol-type tubulin destabilizers, S. cellulosum So ce12 produces sorangicins, potent eubacterial RNA polymerase inhibitors, bactericidal sorangiolides, and the antifungal chivosazoles. To obtain a transposon library of sufficient size suitable for the identification of the presumed biosynthetic gene clusters, an efficient transformation method was developed. We present here the first electroporation protocol for a strain of the genus Sorangium. The transposon library was screened for disorazol-negative mutants. This approach led to the identification of the corresponding trans-acyltransferase core biosynthetic gene cluster together with a region in the chromosome that is likely to be involved in disorazol biosynthesis. A third region in the genome harbors another gene that is presumed to be involved in the regulation of disorazol production. A detailed analysis of the biosynthetic and regulatory genes is presented in this paper.