Pituitary GH3 cells were transfected with different deletion mutants of the human prolactin (hPRL) promoter fused to the CAT reporter gene. The proximal region (-250 to -42) was sufficient to confer stimulation by both thyrotropin-releasing hormone (TRH) and epidermal growth factor (EGF). Further deletion analyses demonstrated the importance of the three proximal Pit-1 binding sites in this response. However, Pit-1 binding oligonucleotides confer neither TRH nor EGF induction to a linked neutral promoter, suggesting that other elements might be involved. We have previously shown that sequence A (-115 to -85) is needed together with Pit-1 binding sites for full cyclic AMP response of hPRL-CAT. Mutation of this sequence strongly affects TRH and EGF induction. On the other hand, three copies of sequence A confer both TRH and EGF response to a linked neutral promoter. In conclusion, although TRH and EGF activate mostly different intracellular pathways, they mediate transcriptional induction of the hPRL promoter via identical cis elements.