We have studied the effect of protein kinase-C activation on the regulation of CRH gene expression in the human hepatoma cell line NPLC/PRF/5 (NPLC), the only cell line known to express the endogenous CRH gene. Incubation of NPLC cells with 100 nM 12-O-tetradecanoyl phorbol 13-acetate (TPA), a phorbol ester that activates protein kinase-C, resulted in a rapid (1-h) and prolonged (72-h) increase in CRH mRNA levels, with the maximum increase of 16-fold observed at 24 h. In addition, TPA treatment increased the size of CRH mRNA by approximately 100 nucleotides. This size increase, which was blocked by protein synthesis inhibitors, occurred within 1 h of TPA addition and lasted at least 8 h, with a return toward the baseline size by 24 h. Structural analysis of CRH mRNA revealed two poly(A) addition sites and, as found in human placenta, multiple transcription start sites. The increase in CRH mRNA size was not due to changes in the sites of either transcription initiation or poly(A) addition, but, rather, to a 3-fold increase in the length of the poly(A) tail itself. The ability of TPA to increase CRH mRNA levels in NPLC cells suggests that the protein kinase-C second messenger pathway may be involved in the physiological regulation of CRH gene expression. Increases in CRH mRNA poly(A) tail length potentially may influence CRH mRNA stability or translatability and, thus, may represent a general mechanism by which the protein kinase-C pathway can influence gene expression.