Through association with CDK1, cyclin B accumulation and destruction govern the G2/M/G1 transitions in eukaryotic cells. To identify CDK1 inactivation-dependent events during late mitosis, we expressed a nondestructible form of cyclin B (cyclin BΔ90) by microinjecting its mRNA into prometaphase normal rat kidney cells. The injection inhibited chromosome decondensation and nuclear envelope formation. Chromosome disjunction occurred normally, but anaphase-like movement persisted until the chromosomes reached the cell periphery, whereupon they often somersaulted and returned to the cell center. Injection of rhodamine-tubulin showed that this movement occurred in the absence of a central anaphase spindle. In 82% of cells cytokinesis was inhibited; the remainder split themselves into two parts in a process reminiscent of Dictyostelium cytofission. In all cells injected, F- actin and myosin II were diffusely localized with no detectable organization at the equator. Our results suggest that a primary effect of CDK1 inactivation is on spindle dynamics that regulate chromosome movement and cytokinesis. Prolonged CDK1 activity may prevent cytokinesis through inhibiting midzone microtubule formation, the behavior of proteins such as TD60, or through the phosphorylation of myosin II regulatory light chain.