Cyclin-dependent kinases (CDKs) are thought to initiate and coordinate cell division processes by sequentially phosphorylating key targets; in most cases these substrates remain unidentified. Using a screen that scores for phosphorylation of proteins, which were translated from pools of cDNA plasmids in vitro, by either phosphoepitope antibody recognition or electrophoretic mobility shifts, we have identified 20 mitotically phosphorylated proteins from Xenopus embryos, 15 of which have sequence similarity to other proteins. Of these proteins, five have previously been shown to be phosphorylated during mitosis (epithelial-microtubule associated protein-115, Oct91, Elongation factor 1gamma, BRG1 and Ribosomal protein L18A), five are related to proteins postulated to have roles in mitosis (epithelial-microtubule associated protein-115, Schizosaccharomyces pombe Cdc5, innercentrosome protein, BRG1 and the RNA helicase WM6), and nine are related to transcription factors (BRG1, negative co-factor 2alpha, Oct91, S. pombe Cdc5, HoxD1, Sox3, Vent2, and two isoforms of Xbr1b). Of 16 substrates tested, 14 can be directly phosphorylated in vitro by the mitotic CDK, cyclin B-Cdc2, although three of these may be physiological substrates of other kinases activated during mitosis. Examination of this broad set of mitotic phosphoproteins has allowed us to draw three conclusions about how the activation of CDKs regulates cell-cycle events. First, Cdc2 itself appears to directly phosphorylate most of the mitotic phosphoproteins. Second, during mitosis most of the substrates are phosphorylated more than once and a number may be targets of multiple kinases, suggesting combinatorial regulation. Third, the large fraction of mitotic phosphoproteins that are presumptive transcription factors, two of which have been previously shown to dissociate from DNA during mitosis, suggests that an important function of mitotic phosphorylation is to strip the chromatin of proteins associated with gene expression.