Biochemical mutants of Actinomyces aureofaciens were obtained after treatment with ultraviolet irradiation or ethyleneimine. A specificity of the mutational process in a series of A. aureofaciens strains was observed, revealing itself mainly in formation of arginine-requiring mutants. Prototroph formation was shown to result from crosses of A. aureofaciens biochemical mutants with either the same or different amino acid requirements. Colonial morphology and antibacterial activity of some prototrophs were studied.