To study the dynamics of interpolar microtubules (iMTs) in Saccharomyces cerevisiae cells, we photobleached a considerable portion of the middle region of anaphase spindles in cells expressing tubulin-green fluorescent protein (GFP) and followed fluorescence recovery at the iMT plus-ends. We found that during anaphase, iMTs show phases of fast growth and shrinkage that are restricted to the iMT plus-ends. Our data indicate that iMT plus-end dynamics are regulated during mitosis, as fluorescence recovery was faster in intermediate anaphase (30 s) compared with long (100 s) and pre-anaphase (80 s) spindles. We also observed that deletion of Cin8, a microtubule-crosslinking kinesin-5 motor protein, reduced the recovery rate in anaphase spindles, indicating that Cin8 contributes to the destabilization of iMT plus-ends. Finally, we show that in cells lacking the midzone organizing protein Ase1, iMTs are highly dynamic and are exchangeable throughout most of their length, indicating that midzone organization is essential for restricting iMT dynamics.