Cyclin-dependent protein kinases (Cdks) in eukaryotic cells work as a key enzyme at various points in the cell cycle. At the onset of S phase, active S-phase Cdks (S-Cdks) are essential for chromosomal DNA replication. Although several replication proteins are phosphorylated in a Cdk-dependent manner, the biological effects of phosphorylation of these proteins on the activation of DNA replication have not been elucidated. Here we show that Sld2 (ref. 4) (also known as Drc1; ref. 5), one of the replication proteins of budding yeast (Saccharomyces cerevisiae), is phosphorylated in S phase in an S-Cdk-dependent manner, and mutant Sld2 lacking all the preferred Cdk phosphorylation sites (All-A) is defective in chromosomal DNA replication. Moreover, the complex that contains, at least, Sld2 and Dpb11 (ref. 6) (the Sld2-Dpb11 complex) is formed predominantly in S phase; the All-A protein is defective in this complex formation. Because this complex is suggested to be essential for chromosomal DNA replication, it seems likely that S-Cdk positively regulates formation of the Sld2-Dpb11 complex and, consequently, chromosomal DNA replication.