The Swi6 transcription factor, required for G1/S-specific gene expression in Saccharomyces cerevisiae, is highly phosphorylated in vivo. Within the limits of resolution of the peptide analysis, the synchrony, and the time intervals tested, serine 160 appears to be the only site of phosphorylation in Swi6 that varies during the cell cycle. Serine 160 resides within a Cdc28 consensus phosphorylation site and its phosphorylation occurs at about the time of maximal transcription of Swi6- and Cdc28-dependent genes containing SCB or MCB elements. However, phosphorylation at this site is not Cdc28-dependent, nor does it control G1/S-specific transcription. The role of the cell cycle-regulated phosphorylation is to control the subcellular localization of Swi6. Phosphorylation of serine 160 persists from late G1 until late M phase, and Swi6 is predominantly cytoplasmic during this time. Aspartate substitution for serine 160 inhibits nuclear localization throughout the cycle. Swi6 enters the nucleus late in M phase and throughout G1, when serine 160 is hypophosphorylated. Alanine substitution at position 160 allows nuclear entry of Swi6 throughout the cell cycle. GFP fusions with the N-terminal one-third of Swi6 display the same cell cycle-regulated localization as Swi6.