Prolactin (PRL) is produced in human thymocytes, T-cells and endometrium. In these extrapituitary tissues, PRL gene transcription is directed by an alternative upstream promoter, and it is thought to act as a locally produced cytokine, with relevance for immune regulation and modulation of T-cell function. We have studied PRL transcriptional regulation in the human T-lymphoblastoid Jurkat cell line transfected with a fragment of the upstream promoter linked to luciferase. A cAMP analogue (cptAMP) increased promoter activity rapidly and dose dependently. This increase was resistant to inhibition by cyclosporin A and thus independent of calcineurin phosphatase (CN). T-cell activation by phorbol myristate acetate (PMA) failed to enhance promoter activity but phytohaemagglutinin (PHA) alone or PHA+PMA increased it, and cptAMP acted in synergy with PMA or PHA to increase it further. H-89, a cAMP-dependent protein kinase A (PKA) inhibitor, inhibited the effect of cptAMP as did transfection with protein kinase inhibitor PKI, an expression vector of the specific inhibitor of PKA. A single point mutation in the CRE (cAMP response element) located at -25 bp in the PRL upstream promoter (TGACGT to TGCCGT) failed to reduce the response to cptAMP, while mutations or deletion of four nucleotides in the CRE to TACTCT diminished the response to cAMP by more than half. We conclude that activity of the human PRL upstream extrapituitary promoter can be induced by activators of T-cells, as well as by a cAMP analogue. The signal is transmitted by PKA and the effect of cAMP is independent of CN. It is partly dependent on an intact proximal CRE motif but a more upstream enhancer may contribute to promoter regulation.