We have studied the dynamics of Aurora B and Survivin during mitosis in living cells, using C-terminal GFP chimeras of the two proteins. These chimeras showed identical localization and behave as bona fide wild type proteins. The mobility of Aurora B-GFP and Survivin-GFP was analyzed by FRAP. The data show that Survivin-GFP, in contrast to Aurora B-GFP, is highly mobile at prometaphase and metaphase. At telophase and cell cleavage, both chimeras are found to be fully immobile. The ablation of Aurora B by siRNA results in a dramatic decrease of the Survivin-GFP mobility. These results demonstrate that Survivin, but not Aurora B, is weakly associated with the centromeric chromatin at prometaphase and metaphase. The weak association of Survivin with centromeric chromatin is dependent on the presence of Aurora B and is not affected by treatment with either nocodazole or taxol. The rapid and conditional interchange between passenger proteins that we show by live imaging indicates that the high affinity interactions demonstrated with in vitro analysis of passenger protein binding are, in fact, static "snapshots" of highly dynamic and regulated in vivo interactions in mitotic cells.