The concentration of mRNA encoding proopiomelanocortin (POMC) was measured in AtT-20/D-16v cells, a clonal pituitary tumor cell line. Treatment of the cells with potassium (20 m M) or veratridine (10 µ M) for 12, 24 and 48 h caused a time-dependent increase in the levels of POMC mRNA which became significant after 24 h. These effects were not seen in the presence of the sodium channel blocker tetrodotoxin (5 µ M). In addition, the calcium channel blocker verapamil (10 µ M) completely abolished the responses to either potassium or veratridine, whereas the calcium channel agonist Bay K 8644 (0.1µ M) potentiated the effect of potassium. Furthermore, the calcium channel blockers verapamil (10 µ M) and nidefipine (1 µ M) significantly decreased not only basal levels of POMC mRNA but also the increase of mRNA levels induced by corticotropin-releasing factor (CRF; 0.1 µ M), 8-bromo-cAMP (1 m M) or cholera toxin (100 ng/ml). The drug-induced alterations in the mRNA POMC levels of the cells were, in each case, associated with similar alterations of immunoreactive β-endorphin in the medium. These results indicate that membrane depolarization to activate sodium channels and calcium channels initiates an entry of calcium ions which triggers POMC gene expression in the AtT-20 cells. Moreover, calcium entry into the cells may exert a tonic stimulatory effect on POMC mRNA under basal conditions and may also contribute to the enhancing effect of CRF or cAMP on POMC mRNA in these cells.