We compared the Sp1 binding activity of Rat2 fibroblasts in nuclear extracts prepared from quiescent cells and cells stimulated with 20% serum. Increased DNA-binding activity was observed in extracts from serum-stimulated cells when an Sp1 oligonucleotide was used as radiolabeled probe in electrophoretic mo-bility shift assays. This increase in Sp1 DNA-binding activity is not due to changes in the amount of Sp1 in the nucleus as shown by immunoblot analysis. The transcriptional activity of a reporter construct con-taining six Sp1 sites upstream of a minimal adenovirus promoter or an Sp1-dependent promoter such as orni-thine decarboxylase (ODC) containing Sp1 sites was enhanced following serum stimulation in transient transfection assays. Dephosphorylation of the nuclear extracts with potato acid phosphatase abolished the Sp1 DNA-binding activity, demonstrating a possible correlation between phosphorylation of Sp1 and DNA-binding activity. These results implicate a po-tential role for Sp1 in mediating signal transduction pathways in response to mitogenic signals.