Entry into mitosis in vertebrate cells is guarded by a checkpoint that can be activated by a variety of insults, including chromosomal damage and disrupting microtubules. This checkpoint acts at the end of interphase to delay cells from entering mitosis, causing cells in prophase to decondense their chromosomes and return to G2 phase. Here, we show that in response to microtubule poisons this "antephase" checkpoint is primarily mediated by the p38 stress kinases and requires the Chfr protein that is absent or inactive in several transformed cell lines and lung tumors. Furthermore, in contrast to previous reports, we find that the checkpoint requires ubiquitylation but not proteasome activity, which is in agreement with the recent demonstration that Chfr conjugates ubiquitin through lysine 63 and not lysine 48.