Cells that are treated long-term with TNFalpha or short-term with TGFalpha together with cycloheximide (CHX) undergo apoptosis. Cell shrinkage and detachment during apoptosis is dependent on actomyosin contractility. Myosin II heavy chain (MHCII) isoforms have shared and distinct functions. Here, we investigated whether the involvement of MHCII isoforms A and B (MHCIIA and MHCIIB, respectively) in cell shrinkage and detachment differs during apoptosis. We show that TNFalpha induces caspase-dependent MHCIIA degradation, whereas MHCIIB levels and association with the cytoskeleton remained virtually unchanged in TtT/GF cells and NIH 3T3 fibroblasts. MHCIIA proteolysis also occurred in fibroblasts that lack MHCIIB when treated with TNFalpha and CHX together. The absence of MHCIIB did not affect cell death rate. However, MHCIIB-/- cells showed more resistance to TNFalpha-induced actin disassembly, cell shrinkage and detachment than wild-type fibroblasts, indicating the participation of MHCIIB in these events. Moreover, inhibition of atypical PKCzeta, which targets MHCIIB but not MHCIIA, blocked TNFalpha-induced shrinkage and detachment in TtT/GF cells and wild-type fibroblasts, but the inhibitory effect was significantly reduced in MHCIIB-/- fibroblasts. TNFalpha treatment increased cytoskeleton-associated myosin light chain (MLC) phosphorylation but did not induce actin cleavage. In conclusion, our results demonstrate that MHCIIB, together with MLC phosphorylation and actin, constitute the actomyosin cytoskeleton that mediates contractility during apoptosis.