Incubation of rifamycin S with washed mycelium of Streptomyces mediterranei gives rifamycin B, rifamycin Y and a new substance named rifamycin L. The structure of the latter has been established as the 4-glycolyl ester of rifamycin SV. The glycolic moieties of rifamycin B and L appear to have the same origin. The biogenetic relationships of these rifamycins are discussed. In this paper we report the isolation and structure determination of rifamycin L, and its biogenetic relationship with the other rifamycins. Moreover the origin of glycolic acid has been investigated. The direct incorporation of some carbon atoms of glucose into the glycolic moiety has been established, but the mechanism of this conversion remains to be elucidated.