N-Acetylisopenicillin N was isolated from the culture broth of Streptomyces tokunonensis sp. nov., and identified from 1H NMR spectroscopy and release of L-alpha-aminoadipic acid by acid hydrolysis. In the course of this study, the specimen of penicillin N isolated from this strain was proved to contain a small amount of isopenicillin N by HPLC.