The mammalian transcription factor LSF (CP2/LBP-1c) binds cellular promoters modulated by cell growth signals. We demonstrate here that LSF-DNA-binding activity is strikingly regulated by induction of cell growth in human peripheral T lymphocytes. Within 15 min of mitogenic stimulation of these cells, the level of LSF-DNA-binding activity increased by a factor of five. The level of LSF protein in the nucleus remained constant throughout this interval. However, a rapid decrease in the electrophoretic mobility of LSF, attributable to phosphorylation, correlated with the increase in DNA-binding activity. pp44 (ERK1) phosphorylated LSF in vitro on the same residue that was phosphorylated in vivo, specifically at amino acid position 291, as indicated by mutant analysis. As direct verification of the causal relationship between phosphorylation and DNA-binding activity, treatment in vitro of LSF with phosphatase both increased the electrophoretic mobility of the protein and decreased LSF-DNA-binding activity. This modulation of LSF-DNA-binding activity as T cells progress from a resting to a replicating state reveals that LSF activity is regulated during cell growth and suggests that LSF regulates growth-responsive promoters.