In the course of studying the sexual reproduction of Phytophthora spp., we isolated aspirochlorine (1), an epidithiodiketopiperazine compound with an unusual 1,3-disulfide linkage, from Aspergillus flavus, which was identical to antibiotic A30641 from A. tamarii. A new antifungal antibiotic, oryzachlorin (2), was reported as an epidithiodiketopiperazine compound with an unelucidated structure, and its 1H NMR spectrum suggested it was a derivative of 1. We reinvestigated the metabolite of the oryzachlorin-producing strain Aspergillus oryzae IAM2613. The strain was cultured in the oryzachlorin production medium (sucrose 5.0%, peptone 0.5%, yeast extract 0.2%, KH2PO4 0.06%, NH4Cl 0.1%, MgSO4·7H2O 0.04%, CaCO3 1.0%) by reciprocal shaking (29°C, 4 days) or surface culture (25-26°C, 20 days). Mycelia were extracted with acetone, the concentrate was combined with the filtrate, and extracted with ethyl acetate. The extract was purified by silica gel column chromatography (CHCl3-MeOH 15:1), benzene-ethyl acetate silica gel chromatography, and Sephadex LH-20 chromatography (methanol), yielding 61 mg of compound 3. All physicochemical and spectroscopic data of 3 were identical to those of aspirochlorine (1). The mass spectrum of oryzachlorin (2) showed m/z 360 (M+) with a chlorine isotope peak at m/z 362, same as 1; its UV spectrum was almost superimposable on that of 1, and its 1H NMR spectrum included all proton signals of 1 (except hydroxyl and amide protons). Aspirochlorine (1) exhibited antimicrobial activity against Candida albicans IAM4888, the indicator organism for oryzachlorin isolation. We conclude that oryzachlorin (2) was not isolated in pure form, and the true active principle of oryzachlorin is aspirochlorine (1). Thus, A. oryzae IAM2613 produces aspirochlorine (1) with an extraordinary epidithiodiketopiperazine structure, as do strains of A. flavus and A. tamarii.