The a naphase p romoting c omplex/c yclosome (APC/C), activated by fzy and fzr, degrades cell cycle proteins that carry RXXL or KEN destruction boxes (d-boxes). APC/C substrates regulate sequential events and must be degraded in the correct order during mitosis and G 1. We studied how d-boxes determine APC/C fzy/APC/C fzr specificity and degradation timing. Cyclin B1 has an RXXL box and is degraded by both APC/C fzy and APC/C fzr; fzy has a KEN box and is degraded by APC/C fzr only. We characterized the degradation of substrates with swapped d-boxes. Cyclin B1 with KEN was degraded by APC/C fzr only. Fzy with RXXL could be degraded by APC/C fzy and APC/C fzr. Interestingly, APC/C fzy-but not APC/C fzr-specific degradation is highly dependent on the location of RXXL. We studied degradation of tagged substrates in real time and observed that APC/C fzr is activated in early G 1. These observations demonstrate how d-box specificities of APC/C fzy and APC/C fzr, and the successive activation of APC/C by fzy and fzr, establish the temporal degradation pattern. Our observations can explain further why some endogenous RXXL substrates are degraded by APC/C fzy, while others are restricted to APC/C fzr.