Piericidins are insecticidal compounds isolated from mycelia of Streptomyces mobaraensis and Streptomyces pactum, toxic to several species of insects, aphids, and mites, with Piericidin A blocking electron transport between NADH dehydrogenase and coenzyme Q. The structures of the piericidin group (An, Bn, Cn, Dn) have been elucidated. In the course of screening for inhibitors of phosphatidylinositol turnover, we isolated a novel antibiotic, piericidin B1 N-oxide, from Streptomyces strain MJ288-OF3. The strain was fermented in seed and fermentation media (sucrose, soybean meal, etc.) at 28°C. The compound was purified via acetone extraction of mycelia, EtOAc extraction, silica gel column chromatography, centrifugal partition chromatography (CPC), Sephadex LH-20 chromatography, and preparative HPLC, yielding 53mg from 6 liters of broth. Piericidin B1 N-oxide is a pale yellow oil soluble in MeOH, EtOAc, and CHCl3 (insoluble in water). Its structure was determined using UV, IR, HRFAB-mass spectrometry (molecular formula C26H39NO5), 1H and 13C NMR, and confirmed by chemical reduction to piericidin B1 (via zinc in CH3COOH). Optical rotation ([α]D25 -4.5°) indicated S-S configurations at C-9 and C-10. It inhibited EGF-stimulated phosphatidylinositol turnover in A431 cells with IC50 1.2μg/ml (stronger than piericidin B1's IC50 5.0μg/ml). Antimicrobial assays showed activity against Gram-positive/negative bacteria and fungi, while piericidin B1 was inactive. Thus, piericidin B1 N-oxide is a new piericidin family member with both phosphatidylinositol turnover inhibitory and antimicrobial activities.