In Saccharomyces cerevisiae, Sic1, an inhibitor of cyclin-dependent kinase, blocks the activity of Cdk1/Clb5,6 (S-Cdk1) kinase required for DNA replication. Deletion of Sic1 causes premature DNA replication from fewer origins, extension of the S phase and inefficient separation of sister chromatids during anaphase. Despite the well documented relevance of Sic1 inhibition of S-Cdk1 for cell cycle control and genome instability, the molecular mechanism by which Sic1 inhibits S-Cdk1 activity remains obscure. In this report, we show that Sic1 is functionally and structurally related to mammalian cyclin-dependent kinase inhibitor (Cki) p27(Kip1) of the Kip/Cip family. A molecular model of the inhibitory domain of Sic1 bound to Cdk2/cyclin A complex suggested that the yeast inhibitor might productively interface with Cdk2/cyclin A mammalian complex. Consistently, Sic1 is able to bind to, and strongly inhibit the kinase activity of, Cdk2/cyclin A mammalian complex. Besides, the comparison of the different inhibitory patterns obtained using histone H1 or GST-pRb as substrates, the last one of which recognizes both the docking site and the catalytic site of Cdk2/cyclin A, offers interesting suggestions on the inhibitory mechanism of Sic1. Finally, overexpression of the KIP1 gene in vivo in Saccharomyces cerevisiae rescues cell cycle-related phenotype of a sic1Delta strain, just as overexpression of the homologous SIC1 gene. Taken together, these findings strongly indicate that budding yeast Sic1 and mammalian p27(Kip1) are functional homologues with a structurally conserved inhibitory domain.