Expression of the gene encoding human inosine- 5'-monophosphate dehydrogenase (IMPDH) type II, an enzyme catalyzing the rate-limiting step in the generation of guanine nucleotides, is increased more than 10-fold in activated peripheral blood T lymphocytes and is required for T cell activation. We have examined the 5'-regulatory sequences that are important for the transcriptional regulation of this gene in T cells. DNase I mapping of genomic DNA identified a hypersensitive element near the transcription initiation site. Fine mapping by in vivo footprinting demonstrated five transcription factor binding sites that are occupied in both resting and activated peripheral blood T lymphocytes; these are tandem CRE motifs, a Sp1 site, an overlapping Egr-1/Sp1 site, and a novel palindromic octamer sequence (POS). The tandem CRE and POS sites are of major functional importance as judged by mutational and electrophoretic mobility shift analyses. These data provide evidence that expression of the human IMPDH type II gene is predominantly regulated by the nuclear factors ATF-2 and an as yet unidentified POS-binding protein. Additional major protein-DNA interactions do not occur within the promoter region after T lymphocyte activation, indicating a requirement for additional protein-protein interactions and/or post-translational modifications of pre-bound transcription factors to account for the observed increase in IMPDH type II gene expression.