After the separation of sister chromatids in anaphase, it is essential that the cell position a cleavage furrow so that it partitions the chromatids into two daughter cells of roughly equal size. The mechanism by which cells position this cleavage furrow remains unknown, although the best current model is that furrows always assemble midway between asters. We used micromanipulation of human cultured cells to produce mitotic heterokaryons with two spindles fused in a V conformation. The majority (15/19) of these cells cleaved along a single plane that transected the two arms of the V at the position where the metaphase plate had been, a result at odds with current views of furrow positioning. However, four cells did form an additional ectopic furrow between the spindle poles at the open end of the V, consistent with the established view. To begin to address the mechanism of furrow assembly, we have begun a detailed study of the properties of the chromosome passenger inner centromere protein (INCENP) in anaphase and telophase cells. We found that INCENP is a very early component of the cleavage furrow, accumulating at the equatorial cortex before any noticeable cortical shape change and before any local accumulation of myosin heavy chain. In mitotic heterokaryons, INCENP was detected in association with spindle midzone microtubules beneath sites of furrowing and was not detected when furrows were absent. A functional role for INCENP in cytokinesis was suggested in experiments where a nearly full-length INCENP was tethered to the centromere. Many cells expressing the chimeric INCENP failed to complete cytokinesis and entered the next cell cycle with daughter cells connected by a large intercellular bridge with a prominent midbody. Together, these results suggest that INCENP has a role in either the assembly or function of the cleavage furrow.