In recent years it has become apparent that the cellular machinery governing cell cycle progression and transcription control are often homologous in yeast and mammalian cells. We and others have previously shown that the SP family of mammalian transcription factors regulates the transcription of a number of genes whose activities are governed by the product of the retinoblastoma (Rb) susceptibility gene, including c-FOS, c-MYC, TGFbeta-1, IGF-II, and c-JUN. To determine whether a similar pathway of transcriptional regulation may function in yeast, we explored the possibility that transcription factors with nucleotide-binding specificities akin to those of the SP family are expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Here we report the detection of novel yeast proteins (S. cerevisiae, p180; S. pombe, p200) that specifically bind Rb-regulated promoter elements in vitro dependent on nucleotides that are also required for binding and trans-activation by SP family members in vivo. Our results indicate that the S. cerevisiae retinoblastoma control element-binding activity 1) requires zinc for association with DNA; 2) does not bind to SCB, MCB, or E2F sites in vitro; 3) is cell cycle-regulated in a SWI6-independent fashion; and 4) maximally stimulates retinoblastoma control element-mediated transcription in early- to mid-S phase. Taken together, these data suggest that p180 may regulate the transcription of a subset of yeast genes whose expression is coincident with the onset and/or progression of DNA replication.