Recent studies have revealed that cell cycle control in G1 or G2 phase is the basis for understanding the mechanism for tumorigenesis. It is therefore likely that inhibitors of G1 and/or G2 progression are candidates for a new type of chemotherapeutic agents. Since the cell cycle regulators such as cyclins and CDKs are highly conserved from yeast to humans, screening for inhibitors of the yeast cell cycle seems to be one of the promising ways leading to identification of mammalian cell cycle inhibitors. The cdc mutants containing temperature-sensitive cell cycle regulators are incapable of progressing through a specific stage of the cell cycle at the nonpermissive temperature. In addition, cdc mutants often show altered sensitivity to agents targeting the cdc gene products at the permissive temperature. We therefore performed a panel screening using several cdc mutants of Schizosaccharomyces pombe (cdc2, cdc13, cdc25, weel) in order to identify compounds to which these cdc mutants showed hyper-sensitivity. In the course of our screening program, we found a strong activity in the broth of a fungus Aspergillus sp. strain 693, which inhibited the proliferation of cdc25, cdc13, and weel but did not produce any visible inhibitory zone on the wild-type or other cdc strains in the paper disc assay. The active compound was purified and identified as preussin (C21H35NO) via spectroscopic analyses, which was previously reported as an antifungal antibiotic. Minimal inhibitory concentrations (MICs) of preussin against S. pombe cdc mutants were determined by a conventional agar dilution method. The MICs of preussin for cdc13, cdc25, weel, and weel disrupted strains were 24 ng/ml, 12 ng/ml, 391 ng/ml, and 196 ng/ml, respectively, which were 100 to 1000-fold more sensitive compared with wild-type S. pombe (MIC 12.5 μg/ml). To determine the direct target of preussin, we tested its effect on Cdc2 kinase, Cdc25 phosphatase, and Weel kinase using recombinant proteins. Preussin did not inhibit their activity in vitro at high concentrations. Preussin also caused growth inhibition of mammalian cells. The IC50 of preussin for normal rat fibroblast 3Y1 cells was 5 μg/ml. Flow cytometry analysis showed that preussin inhibits G1 progression of the mammalian cell cycle in a relatively narrow concentration range and causes a cytotoxic effect at a high concentration. The relationship between selective growth inhibition in S. pombe cdc mutants and the G1 arrest in mammalian cells is unclear. Further studies are needed for elucidation of molecular mechanism by which preussin induces selective growth inhibition of S. pombe cdc mutants and G1 arrest in mammalian cells.