First described in Arabidopsis thaliana, Tousled-like kinases (Tlks) are highly conserved in both plants and animals. In plants, Tousled kinase is essential for proper flower and leaf development, but no direct functional link to any other plant gene product has yet been established. Likewise, the role of Tlks in animals is unknown. In human cells, two structurally similar Tlks, Tlk1 and Tlk2, were recently shown to be cell cycle-regulated kinases with maximal activities during S phase. Here, we report the identification of two human homologs of the Drosophila chromatin assembly factor Asf1 (anti-silencing function 1) as physiological substrates of Tlks. We show that human Asf1 proteins are phosphorylated by Tlks both in vivo and in vitro. Furthermore, Asf1 proteins are phosphorylated during S phase, when Tlks are maximally active. Conversely, Asf1 proteins are dephosphorylated upon the activation of the DNA replication checkpoint, concomitant with the rapid inactivation of Tlks. These data indicate that Tlk family members regulate chromatin assembly during DNA replication, and they suggest a plausible explanation for the pleiotropic developmental defects of plant tousled mutants.