Spindle microtubules (MTs) in PtK1 cells, fixed at stages from metaphase to telophase, have been reconstructed using serial sections, electron microscopy, and computer image processing. We have studied the class of MTs that form an interdigitating system connecting the two spindle poles (interpolar MTs or ipMTs) and their relationship to the spindle MTs that attach to kinetochores (kMTs). Viewed in cross section, the ipMTs cluster with antiparallel near neighbors throughout mitosis; this bundling becomes much more pronounced as anaphase proceeds. While the minus ends of most kMTs are near the poles, those of the ipMTs are spread over half of the spindle length, with at least 50% lying > 1.5 microns from the poles. Longitudinal views of the ipMT bundles demonstrate a major rearrangement of their plus ends between mid- and late anaphase B. However, the minus ends of these MTs do not move appreciably farther from the spindle midplane, suggesting that sliding of these MTs contributes little to anaphase B. The minus ends of ipMTs are markedly clustered in the bundles of kMTs throughout anaphase A. These ends lie close to kMTs much more frequently than would be expected by chance, suggesting a specific interaction. As sister kinetochores separate and kMTs shorten, the minus ends of the kMTs remain associated with the spindle poles, but the minus ends of many ipMTs are released from the kMT bundles, allowing the spindle pole and the kMTs to move away from the ipMTs as the spindle elongates.