Protoplast regeneration of Amycolatopsis orientalis subsp. eremomycini producing eremomycin leads to the change of cultural and morphological properties as well as synthesis of secondary metabolites. Formation of plus-variants with enchanced antibiotic production was promoted by UV-irradiation of protoplasts. These plus-variants can be successfully used for repeating protoplasting--UV-irradiation of protoplasts with further increasing of the strain productivity. Finally activity of the initial A. orientalis culture was increased 7-8 times. Proposed method is recommended for the improvement of actinomycetes strains producing antibiotics especially in the case of cultures with poor sporulation.