Esperamicins, a family of extremely potent compounds showing a broad spectrum of antimicrobial and antitumor activities in murine models, have been identified in cultures of Actinomadura verrucosospora ATCC 39334. The isolation and structure elucidation of esperamicins A₁, A₁b, and A₂ have been reported. While developing a chemically defined medium (DF-15) for more efficient incorporation of labelled precursors into esp A₁ during biosynthetic studies, two new esperamicin analogs were detected. This paper reports the production, isolation, structure determination, and antitumor activity of these analogs. Shifting fermentation of A. verrucosospora ATCC 39334 from complex medium H946 to defined medium DF-15 (with sucrose as the sole carbon source and ammonium sulfate as the sole nitrogen source) yielded a significantly different esperamicin production profile. The two new analogs were identified as esp A₁c (later determined to be the same as FR-900406 reported by Fujisawa Pharm. Co. Ltd.) and esp A₂c (a novel analog not previously reported). Antitumor activity assays against P388 leukemia in CDF₁ mice showed that esp A₁c exhibited significant activity (e.g., 175% T/C at a dose of 0.016 mg/kg, ip) and esp A₂c had moderate activity (145% T/C at 0.016 mg/kg, ip).