AbstractRifamycins are primarily produced by Gram-positive bacteriumAmycolatopsis mediterranei, which belongs to the order Actinomycetales. These antibiotics, apart from their application against pathogens of tuberculosis and leprosy, have also been found to be effective against several other pathogens includingMycobacterium aviumandPneumococcus. Because of the importance of rifamycin, the producer strainA. mediterraneihas been genetically manipulated since 1957 in order to develop a strain that can either produce larger amounts of rifamycin or derivatives of rifamycin. In this article, the importance of the producer strain, traditional methods (mutations and recombination) of strain improvement, their limitations, and the development of a cloning vector and transformation methods that have made recombinant DNA techniques accessible for genetic manipulations ofA. mediterraneiare discussed.