Abstract: The CDC40 (PRP17) gene of S. cerevisiae encodes a splicing factor required for multiple events in the mitotic and meiotic cell cycles, linking splicing with cell cycle control. cdc40 mutants exhibit a delayed G(1)/S transition, progress slowly through S-phase and arrest at a restrictive temperature in the G(2) phase. In addition, they are hypersensitive to genotoxic agents such as methylmethane sulfonate (MMS) and Hydroxyurea (HU). CDC40 has been suggested to control cell cycle through splicing of intron-containing pre-mRNAs that encode proteins important for cell cycle progression. We screened a cDNA overexpression library and isolated cDNAs that specifically suppress the HU/MMS-sensitivity of cdc40 mutants. Most of these cDNAs surprisingly encode chaperones, translation initiation factors and glycolytic enzymes, and none of them is encoded by an intron-containing gene. Interestingly, the cDNAs suppress the G(1)/S transition delay of cdc40 cells, which is exacerbated by HU, suggesting that cdc40 mutants are HU/MMS-sensitive due to their S-phase entry defect. A role of Cdc40p in passage through G(1)/S (START) is further supported by the enhanced temperature sensitivity and G(1)/S transition phenotype of a cdc40 strain lacking the G(1) cyclin, Cln2p. We provide evidence that the mechanism of suppression by the isolated cDNAs does not (at least solely) involve up-regulation of the known positive START regulators CLN2, CLN3, DCR2 and GID8, or of the large and small essential ribonucleotide reductase (RNR) subunits, RNR1 and RNR2. Finally, we discuss possible mechanisms of suppression by the cDNAs that imply cell cycle regulation by apparently unrelated processes, such as splicing, translation initiation and glycolysis.