The GAGA box of the rat serine protease inhibitor 2 (spi 2) genes not only acts as a basal promoter element, but also mediates transcriptional activation by growth hormone and interleukin-6. The GAGA box is separated from the TATA box by only 12 bp, and this close association is required for efficient transcription. Hence, the GAGA box may influence transcription efficiency through interactions between GAGA box binding proteins and some components of the RNA polymerase II complex. Here we report the cloning of two GAGA box-binding proteins termed p38 and p40, that belong to the type A/B heterogeneous nuclear ribonucleoprotein subgroup. GAGA box mutations that diminish the affinity for p38 and p40 decrease basal and GH-induced reporter gene expression. Furthermore, nuclear extracts depleted of p38 and p40 can no longer support GAGA box-dependent in vitro transcription. Therefore, two polypeptides previously assigned to a family of RNA processing proteins also act as DNA-binding, promoter-specific transcription factors.