During vertebrate cytokinesis it is thought that contractile ring constriction is driven by nonmuscle myosin II (NM II) translocation of antiparallel actin-filaments, similar to muscle contraction. Here we report in vivo, in situ and in vitro observations that challenge this hypothesis. NM II-B is essential for normal cardiac myocyte development. Ablation of NM II-B in mice resulted in defects in cardiac myocyte cytokinesis. Surprisingly, expression of mutant NM II-B R709C that cannot translocate actin filaments and has a substantially diminished MgATPase activity, in place of wild-type NM II-B, successfully rescues multinucleation in NM II-B ablated cardiomyocytes in mouse hearts. Graded siRNA knockdown of NM II-B in cultured COS-7 cells reveals that the amount of NM II limits contractile ring constriction. Time-lapse analyses show that both the rate and extent of ring constriction depends on the level of NM II expression. In addition expression of motor-impaired mutant NM IIs (NM II-B R709C, NM II-A N93K and NM II-A R234A) restores contractile ring constriction in COS-7 cells depleted of NM II-B, even though these mutant NM IIs are incapable of translocating actin-filaments. However contractile ring constriction is blocked by blebbistatin which keeps NM II in the weakly bound (to actin) state, in cells expressing either wild-type or mutant NM IIs. These results support a role for NM II in generating tension but not translocating actin-filaments during contractile ring constriction. This role is substantiated by in vitro transient kinetic experiments with baculovirus-expressed NM II proteins using stopped-flow analyses. The mechanochemical properties of mutant NM II-B R709C show extremely high affinity for actin, despite loss of actin translocation. Under loaded conditions, mutant NM II exhibits prolonged strong actin attachment during which a single mechanoenzymatic cycle spans most of the time of cytokinesis. This prolonged attachment promotes simultaneous binding of essentially all NM II heads to actin, thereby increasing tension generation and resisting expansion of the ring and cell cortex, but further preventing translocation of actin-filaments.