Orderly progression through the cell cycle is essential for the correct development and survival of all eukaryotes. DNA replication during S phase and cell division at mitosis are driven by a conserved family of kinases, the cyclin-dependent kinases (CDKs). CDKs are dowregulated by a number of different mechanisms, such as inhibitory phosphorylation, cyclin proteolysis and direct inhibition by cyclin-dependent kinase inhibitor (CKI) proteins. In Drosophila, the Roughex (Rux) gene product prevents premature S phases by inhibiting CycA-dependent kinase activity. Here, it is shown that Rux fulfills all the criteria that define proteins as CKIs. rux overexpression inhibits mitosis. Rux physically associates with CycA and CycB, inhibits Cdk1 dependent kinase activity and prevents Cdk1 activation. Rux does not inhibit Cdk2/CycE and overexpression of rux does not prevent CycE dependent S phases. Thus, Rux is a Drosophila CKI specific for mitotic cyclins. This is the first such CKI to be described in a multicellular organism. In addition to G1 maintenance Rux also plays a role in mitotic exit. Observations of fixed and living embryos show that metaphase is significantly longer in rux mutants than in wild-type embryos. In addition, Rux overexpression is sufficient to drive cells experimentally arrested in metaphase into interphase. Furthermore, rux mutant embryos are impaired in their ability to overcome a transient metaphase arrest induced by expression of a stable CycA. Rux has numerous functional similarities with Sic1. While these proteins share no sequence imilarity, Sic1 inhibits mitotic Cdk1-cyclin complexes from Drosophila in vitro and in vivo. Rux is stable during most of the cell cycle, with the exception of the G1 to S phase transition where it is destroyed by the proteasome. Rux destruction relies on a PEST sequence in the C-terminus of the protein. Based on the results described above the following model is proposed for Rux function; Rux is a Drosophila CKI specific for mitotic cyclins that inhibits Cdk1/CycA activity during metaphase and maintains this activity at a low level during G1. Rux is destroyed by the proteasome at transition into S phase, liberating CycA to perform S phase functions.