Sp1 sites can mediate growth/cell cycle induction of dihydrofolate reductase in late G1 (Jensen, D. E., Black, A. R. Swick, A. G., and Azizkhan, J. C. (1997) J. Cell. Biochem. 67, 24-31). To investigate mechanisms underlying this induction, effects of serum stimulation on regulation of Sp1 were examined. In Balb/c 3T3 cells, serum stimulation did not affect Sp1 synthesis or the relative binding of Sp1 family members to DNA; however, it did result in a rapid, approximately 2-fold increase in Sp1 levels and an approximately 3-fold increase in specific Sp1 phosphorylation in mid-G1. In normal human diploid fibroblasts, serum stimulation also increased Sp1 phosphorylation in mid-G1 but did not affect Sp1 levels. Therefore, Sp1 phosphorylation is regulated in a growth/cell cycle-dependent manner which correlates temporally with induction of dihydrofolate reductase transcription. Further studies revealed a kinase activity specifically associated with Sp1 in a growth-regulated manner. This activity is distinct from purified kinases previously shown to phosphorylate Sp1 in vitro and phosphorylates Sp1 between amino acids 612 and 678 in its C terminus, a region also phosphorylated in mid-G1 in vivo. Therefore, this study indicates that phosphorylation of the C terminus of Sp1 may play a role in the cell cycle regulation of its transcriptional activity.