The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction following DNA damage. To gain further insight into the processes regulating E2F1 protein stability, we focused our investigation on the mechanisms responsible for regulating E2F1 turnover. Here we show that E2F1 is a substrate of the anaphase promoting complex or cyclosome (APC/C), a ubiquitin ligase that plays an important role in cell cycle progression. Ectopic expression of the APC/C activators Cdh1 and Cdc20 reduced the levels of co-expressed E2F-1 protein. Co-expression of DP1 with E2F1 blocked APC/C-induced E2F1 degradation, suggesting that the E2F1/DP1 heterodimer is protected from APC/C regulation. Following Cdc20 knockdown, E2F1 levels increased and remained stable in extracts over a time course, indicating that APC/C(Cdc20) is a primary regulator of E2F1 stability in vivo. Moreover, cell synchronization experiments showed that siRNA directed against Cdc20 induced an accumulation of E2F1 protein in prometaphase cells. These data suggest that APC/C(Cdc20) specifically targets E2F1 for degradation in early mitosis and reveal a novel mechanism for limiting free E2F1 levels in cells, failure of which may compromise cell survival and/or homeostasis.