Resting human peripheral blood T cells synethesize proteins at very low rates and contain very low levels of eIF-2 alpha mRNA. During mitogenic activation, the level of eIF-2 alpha mRNA increases at least 50-fold, an effect thought to be due primarily to intranuclear stabilization of the primary transcript. Analysis of sequences (+447 to +457) within the first intron revealed a region with homology to the initiator (Inr) sequence first described by Smale and Baltimore. This Inr element is positioned 450 bases downstreams of the eIF-2 alpha promoter and is oriented to generate an overlapping antisense transcript. Deletion or mutation of the Inr element results in a reproducible 5-8 fold increase in the activity of an eIF-2 alpha promoter driven CAT reporter gene and a corresponding 2.5 fold decrease in activity of an antisense driven luciferase report gene in vivo in 293 cells. In vitro transcription analysis also reveals antisense transcripts which depend on an intact Inr element and whose 5' ends map to sequences surrounding the Inr consensus sequence. By DNase I footprint analysis and electrophoretic mobility shift assay, we have found a potential cis-regulatory sequence immediately adjacent to the Inr element between +457 and +474. In addition to conferring protection against DNase I, binding of a 43 kD factor also generates hypersensitive sites directly over the Inr element. The antisense orientation of the Inr element with the first intron of the eIF-2 alpha gene suggests a role for the antisense transcript in the regulation of expression of eIF-2 alpha. The role of antisense transcripts and the role of the 43 kD protein in regulation of antisense transcription are currently under investigation.