The biological active compound rishirilide B is produced by Streptomyces bottropensis. The cosmid cos4 contains the complete rishirilide B biosynthesis gene cluster. Its heterologous expression in the host Streptomyces albus J1074 led to the production of rishirilide B as a major compound and to small amounts of rishirilide A, rishirilide D and lupinacidin A. In order to gain more insights into the biosynthesis, gene inactivation experiments and gene expression experiments were carried out. This study lays the focus on the functional elucidation of the genes involved in the early biosynthetic pathway. A total of eight genes were deleted and six gene cassettes were generated. Rishirilide production was not strongly affected by mutations in rslO2, rslO6 and rslH. The deletion of rslK4 and rslO3 led to the formation of polyketides with novel structures. These results indicated that RslK4 and RslO3 are involved in the generation or selection of the starter unit for rishirilide biosynthesis. In the rslO10 mutant strain, two novel compounds were detected, which were also produced by a strain containing solely the genes rslK1, rslK2, rslK3, rslK4, and rslA. rslO1 and rslO4 mutants predominately produce galvaquinones. Therefore, the ketoreductase RslO10 is involved in an early step of rishirilide biosynthesis and the oxygenases RslO1 and RslO4 are most probably acting on an anthracene moiety. This study led to the functional elucidation of several genes of the rishirilide pathway, including rslK4, which is involved in selecting the unusual starter unit for polyketide synthesis.