Previously, we isolated three indole alkaloids, roquefortine A, B and C, from the cultures of Penicillium roqueforti, and made clear the structures of those alkaloids. Recently, we have isolated the unknown indole alkaloid "Z" from the cultures of the same Penicillium mold. This alkaloid "Z" was obtained in yields of 50 mg together with 600 mg of roquefortine C from the mycelium and culture filtrate when a selected strain was grown at 30°C for 20 days in mannitol-glucose-ammonium succinate medium. The alkaloid "Z" crystallized from acetonitrile or methanol in colorless prisms; mp 153~154°C, [α]₁₈ᴰ -370° (c=0.23, pyr.). Its molecular formula was C₂₂H₂₅N₅O₂. IR-spectrum showed bands for a diketopiperazine unit (3200 and 1660 cm⁻¹, no amide II band). NMR-spectrum lacked the vinyl proton singlet of roquefortine C's dehydrohistidine side chain and had characteristic double doublets. MS-spectrum fragmentation (loss of C₅H₉ to give m/e 322 base peak) supported it as a dihydro-derivative of roquefortine C. Chemical and spectro-chemical properties, thin-layer chromatography, and mixed melting point confirmed "Z" was identical to isomer-1 of dihydroroquefortine C (prepared by reducing roquefortine C with Zn in acetic acid). Thus, alkaloid "Z" is a new indole alkaloid with the structure of dihydroroquefortine C isomer-1, named roquefortine D.