We have described previously that, during S-phase, human DNA ligase I is phosphorylated on Ser66, a casein kinase II site. Here we investigate the phosphorylation status of DNA ligase I during the cell cycle by gel shift analysis and electrospray mass spectrometry. We show that three residues (Ser51, Ser76, and Ser91), which are part of cyclin-dependent kinase sites, are phosphorylated in a cell cycle-dependent manner. Phosphorylation of Ser91 occurs at G 1/S transition and depends on a cyclin binding site in the C-terminal part of the protein. This modification is required for the ensuing phosphorylation of Ser76 detectable in G2/M extracts. The substitution of serines at positions 51, 66, 76, and 91 with aspartic acid to mimic the phosphorylated enzyme hampers the association of DNA ligase I with the replication foci. We suggest that the phosphorylation of DNA ligase I and possibly other replicative enzymes is part of the mechanism that directs the disassembly of the replication machinery at the completion of S-phase.