Abstract During mitotic arrest induced by paclitaxel, most of the mitochondrial Bcl-2 is phosphorylated. This mitotic arrest is transient; exit from mitosis, due to mitotic slippage, occurs and Bcl-2 is rapidly dephosphorylated. In the present study, we characterized PP1 as the cytosolic phosphatase involved in Bcl-2 dephosphorylation. When mitochondria and cytosol prepared from mitotic arrested cells were incubated in vitro, the proportion of phosphorylated forms of Bcl-2 in mitochondria remained unchanged. In contrast, cytosol prepared from cells during mitotic slippage led to a dose-dependent loss of phosphorylated forms of Bcl-2. Depletion of these cytosol extracts by microcystin–Sepharose maintained Bcl-2 phosphorylated forms, indicating that this cytosol possessed phosphatase activity. Furthermore, the dephosphorylation of Bcl-2 by cytosol prepared from cells exiting mitotic block was inhibited by okadaic acid, at a dose known to inhibit PP1, and by inhibitor 2, a specific inhibitor of PP1 and by immunodepletion of PP1. Finally, we showed that PP1 is associated with mitochondrial Bcl-2 in vivo. Taken together, these results demonstrate that PP1 is directly involved in Bcl-2 dephosphorylation during mitotic slippage.