The nucleus of the mature human neutrophil is segmented into three to five interconnected lobes. The physiological purpose of this segmentation is unknown, as is the mechanism by which the lobes are formed during differentiation. Using video observation of migrating human neutrophils simultaneously illuminated for fluorescence and phase-contrast microscopy, we analyzed nuclear movements with respect to cell shape changes. The number of nuclear lobes and their relative size remained constant during observation (up to 1 h). The thin connecting segments between the lobes elongated and attenuated extensively but never separated. Electron microscopic analysis of neutrophil nuclei revealed no specialized nuclear or cytoplasmic structures in the vicinity of connecting segments. With fluorescence in situ hybridization of whole chromosome probes, we determined that chromosomes are randomly distributed among neutrophil nuclear lobes. HL60 cells are a human myelocytic line that, with retinoic acid treatment, segment their nuclei and differentiate into neutrophil-like cells over several days. Using a rapidly responding variant line termed HL60/S4 (Cancer Res. 52, 949-954), we found that segmentation could be induced within 24 h. We tested the role of cytoskeletal elements in the process of nuclear segmentation. Neither the microtubule inhibitor nocodazole nor the microfilament inhibitor cytochalasin D prevented nuclear segmentation. Together, our studies suggest that nuclear lobes in neutrophils are relatively stable structures that are not generated by microtubule- or microfilament-dependent forces.