Although chromatin condensation is one of the hallmarks of apoptosis, its relationship with DNA fragmentation has been controversial. We show here that apoptotic chromatin condensation is regulated by nucleoplasmin, a protein that decondenses sperm chromatin during male pronuclear assembly. In Xenopus egg extracts, nucleoplasmin is tyrosine-dephosphorylated during apoptosis. This dephosphorylation inactivates the chromatin decondensation activity of nucleoplasmin and leads to its exclusion from the chromatin. Inhibition of tyrosine dephosphorylation prevents apoptotic chromatin condensation but not DNA fragmentation. Studies with mutant proteins indicate that dephosphorylation of nucleoplasmin at Tyr-124 regulates chromatin condensation through changes in the interaction of nucleoplasmin with chromatin and the loss of its chromatin decondensation activity. These results show that chromatin condensation and DNA fragmentation are independent processes.