Illudin S primarily and specificially inhibited RNA synthesis at a concentration of 1-10ng/ml of culture medium in the early period of G(1)-S phase transition as well as in S phase in a growing medium. DNA synthesis in S phase and cell proliferation were inhibited as secondary events. The inhibitory effects on RNA synthesis were only manifested in intact cell systems, not in cell-free ones. DNA replication in a nucleotide-permeable cell system was completely insensitive to Illudin S. Protein synthesis in intact cell systems was not inhibited by Illudin S. Illudin S should prove useful in the analysis of the mechanism of G(l)-S phase transition in the cell cycle; especially in the elucidation of specific kinds of RNA required for initiation of DNA replication.