Regulated degradation plays a key role in setting the level of many factors that govern cell cycle progression. In Drosophila, the largest subunit of the origin recognition complex protein 1 (ORC1) is degraded at the end of M phase and throughout much of G1 by anaphase-promoting complexes (APC) activated by Fzr/Cdh1. We show here that none of the previously identified APC motifs targets ORC1 for degradation. Instead, a novel sequence, the O-box, is necessary and sufficient to direct Fzr/Cdh1-dependent polyubiquitylation in vitro and degradation in vivo. The O-box is similar to but distinct from the well characterized D-box. Finally, we show that O-box motifs in two other proteins, Drosophila Abnormal Spindle and Schizosaccharomyces pombe Cut2, contribute to Cdh1-dependent polyubiquitylation in vitro, suggesting that the O-box may mediate degradation of a variety of cell cycle factors.