Skyrin was extracted from Penicillium islandicum cultivated on synthetic Czapek-Dox medium. A preliminary experiment in Penicillium islandium using [^(14)C]-acetate proved the incorporation of [^(14)C] into skyrin. In the cell-free system using the fungus [³H]-emodin and [³H]-emodinanthrone were shown to give some reliable evidences far the biosynthesis of skyrin. The incorporation of [³H]-emodin was less significant than that of [³H]-emodinanthrone. As a result of the cell-free experiment, the biosynthetic pathway of skyrin by the fungus was suggested as follows: emodinanthrone→emodin→ skyrin.