Exit from mitosis in eukaryotic cells is regulated by the cyclosome (also called anaphase promoting complex or APC), a multisubunit ubiquitin ligase that acts on mitotic cyclins. Previous studies in a cell-free system from clam oocytes have shown that the activation of the cyclosome at the end of mitosis involves its phosphorylation by protein kinase Cdk1/cyclin B. Genetic and biochemical studies have furthermore indicated that cyclosome activity also requires a WD-40 repeat containing protein called Fizzy (FZY) or Cdc20. It has been suggested [Fang et al. (1998) Mol. Cell 2, 163-171] that in the presence of FZY, the phosphorylation of the cyclosome is not critical for its activation. By contrast, we find that the activity of the interphase, non-phosphorylated form of the cyclosome from clam embryos is not stimulated by FZY to a significant extent. However, when interphase cyclosome is first incubated with protein kinase Cdk1/cyclin B, the subsequent supplementation of FZY greatly stimulates its cyclin-ubiquitin ligase activity. Furthermore, phosphatase treatment of purified mitotic cyclosome prevents its stimulation by FZY, a process that can be reversed by the action of protein kinase Cdk1/cyclin B. We conclude that in the early embryonic cell cycles, the primary event in the activation of the cyclosome at the end of mitosis is its Cdk1-dependent phosphorylation and activation by FZY takes place in a subsequent process.