We show here that the fission yeast gene products Cut9 and Nuc2 are the subunits of the 20S complex, the putative APC (anaphase promoting complex)/cyclosome which contains ubiquitin ligase activity required for cyclin and Cut2 destruction. The assembly of Cut9 into the 20S complex requires functional Nuc2, and vice versa. The size of fission yeast APC/cyclosome is similar to that of higher eukaryotes, but differs greatly from that (36S) of budding yeast. The 20S complex is present in cells arrested at different stages of the cell cycle, and becomes slightly heavier in mitosis than interphase. Cut9 in the 20S complex is hyperphosphorylated specifically at the time of metaphase. The truncated forms of Cut9 block entry into mitosis, however. The 20S assembly impaired in the cut9 mutant can be restored by elevating the level of a novel gene product Hcnl, similar to budding yeast Cdc26. Furthermore, deletion of protein kinase PKA (Pkal) suppresses the phenotype of the cut9 mutation and reduces phosphorylation of Cut9. In contrast, PP1 (Dis2) phosphatase mutation shows the reverse effect on the phenotype of cut9. The Cut9 subunit is likely to be a target for regulating APC/ cyclosome function through protein-protein interactions and phosphorylation.