Cytokinesis partitions the cytoplasm of dividing eukaryotic cells. In higher plants, a dynamic microtubule array--phragmoplast--mediates the formation of the partitioning membrane--cell plate--in a centrifugal fashion. This phragmoplast dynamic involves microtubule-associated proteins. Mutations in a novel Arabidopsis gene RUNKEL (RUK) result in cytokinesis defects caused by abnormal phragmoplast organization and arrested cell plate expansion. RUK encodes an essential cell-cycle-regulated 152 kDa protein with a putative serine/threonine kinase domain and a large microtubule-binding domain, both of which are largely conserved in uncharacterized proteins from protozoa, plants, and animals. RUK directly bound to microtubules in vitro and colocalized with mitotic preprophase band, spindle, and phragmoplast in vivo. An engineered RUK fusion protein that was degraded before telophase did not rescue the ruk mutant phenotype, demonstrating RUK action during cytokinesis. Both microtubule-binding domain and putative kinase domain were essential for RUK function. Surprisingly, RUK did not show kinase activity in vitro, and transgenically expressed "kinase-dead" RUK rescued the seedling lethality of ruk mutants. Our results suggest that RUK plays a regulatory, rather than catalytic, role in phragmoplast microtubule organization during cell plate expansion in cytokinesis.