Efficient transcription of SV40 early genes requires transcription factor Spl. Here, we report that SV40 infection induces Spl phosphorylation. While characterizing this modification, we discovered that Spl becomes quantitatively phosphorylated in an in vitro transcription extract. Multiple processive phosphoryiation of Spl depends on binding of Spl to GC boxcontaining DNA. Cell fractionation and column chromatography reveal that the Spl kinase is a nuclear DNA binding protein that corresponds to a pmviously identified DNA-dependent protein kinase. Because only some frans-activators are phosphorylated by this kinase, Spl belongs to a specific subgroup of factors that an? phosphoryiated upon binding to promoter sequences. Finally, efficient phosphorylation of Spl requires both a functional DNA binding domain and a region containing the transcriptional activation domains. Coupling of phosphorylation to DNA binding may represent a novel mechanism for regulating transcriptional initiation;