In the fission yeast Schizosaccharomyces pombe, the execution of Start requires the activity of the Cdc2 protein kinase and the Cdc10/Sct1 transcription complex. The loss of any of these genes leads to G1 arrest and activation of the mating pathway under appropriate conditions. We have undertaken a genetic and biochemical analysis of these genes and their protein products to elucidate the molecular mechanism that governs the regulation of Start. We demonstrate that serine-196 of Cdc10 is phosphorylated in vivo and provide evidence that suggests that phosphorylation of this residue is required for Cdc10 function. Substitution of serine-196 of Cdc10 with alanine (Cdc10 S196A) leads to inactivation of Cdc10. We show that Cdc10 S196A is incapable of associating with Sct1 to form a heteromeric complex, whereas substitution of this serine with aspartic acid (S196D) restores DNA-binding activity by allowing Cdc10 to associate with Sct1. Furthermore, we demonstrate that Cdc2 activity is required for the formation of the heteromeric Sct1/Cdc10 transcription complex and that the Cdc10 S196D mutation alleviates this requirement. We thus provide biochemical evidence to demonstrate one mechanism by which the Cdc2 protein kinase may regulate Start in the fission yeast cell cycle.