The mechanism by which MPF induces nuclear lamin disassembly and nuclear envelope breakdown during mitosis was studied in a frog egg extract in which the transition from interphase to mitosis can be induced by the addition of MPF. Bacterially expressed human nuclear lamin C, assembled in vitro into filaments, showed increased phosphorylation on specific sites in the extract in response to MPF. Phosphorylation was accompanied by disassembly of the lamin filaments. We determined the sequences of the sites phosphorylated both in the presence and absence of MPF. The sequence data suggest that multiple protein kinases act on the lamins, and S6 kinase II was identified as one potentially important lamin kinase.