Benadrostin, a new inhibitor of poly(ADP-ribose) synthetase, has been isolated from the culture broth of Streptomyces flavovirens MH499-O'F1. The structure of benadrostin was defined as 8-hydroxy-2H-1,3-benzoxazine-2,4-dione by an analysis of spectral properties and chemical studies of benadrostin and its derivatives.