In cultures of the epithelial cell lines, PtK2 and LLC-PK, some cells assume unusually large flattened morphologies and, during cell division, produce unusual cleavage furrows. We have microinjected some of these large cells with fluorescent actin and myosin probes to determine how the cell's shape and the position of its mitotic spindle affect the deposition of actin and myosin in the forming cleavage furrow. In cells with two spindles, contractile proteins were recruited not only to the cortex bordering the former metaphase plates but also to the cortex midway between each pair of adjacent nondaughter poles or centrosomes. The furrowing between adjacent poles seen in these cultured epithelial cells conformed to the furrows seen when echinoderm eggs were manipulated into a torus shape so that the poles of two mitotic spindles were adjacent to one another [Rappaport, 1961]. The recruitment of contractile proteins and the formation of furrows between adjacent centrosomes was a function of the distances between them. When adjacent centrosomes were positioned too close together neither contractile protein recruitment nor furrow formation occurred. If a normal-sized spindle was present in a very large cell, fibers of contractile protein assembled in the cortex above the former metaphase plate but they did not extend to the cell periphery, resulting in an inhibition of cytokinesis. In all injected cells, the recruitment of actin and myosin to the cell surfaces could first be detected at mid-anaphase before there was any visible sign of furrowing. Our results suggest that vertebrate cells share common mechanisms for the establishment of the cleavage furrow with echinoderm cells. The results are consistent with a model in which (1) the positions of the centrosomes and their linearly connected microtubules determine the position for the assembly of the cleavage furrow, and (2) the signal arrives at the surface within a few minutes after the initiation of anaphase. We speculate that an interaction of the ends of microtubules from adjacent centrosomes with the cell surface promotes a clustering of integral membrane protein(s) that interact with and target contractile proteins to a position midway between centrosomes where furrowing occurs.