Monolayer cultures of the GH3 clone of functional rat pituitary cells have been used to study the short-term effects of cations and colchicine on the release of prolactin (PRL) and growth hormone (GH). Intracellular and extracellular concentrations of the hormones were measured by immunological methods. Increased extracellular concentrations of potassium (50 HIM) enhanced the release of both PRL and GH while the same concentration of sodium had no effect. Reduced or elevated levels of extracellular calcium had no acute (3 hr) effect on hormone release. Similarly, increased extracellular magnesium (10 HIM) did not affect hormone release. High potassium, but not high sodium, calcium or magnesium, had a hormone-releasing effect on cells in which the synthesis of PRL and GH had been stimulated by thyrotropin-releasing hormone or hydrocortisone, respectively. The hormone- releasing effect of high potassium treatment did not occur if the culture medium was low in calcium. Colchicine (5 X 10-6 M ) inhibited acutely (3 hr) the release of both PRL and GH. This effect of colchicine was overcome by an increase in extracellular calcium. The effect of colchicine was also antagonized by a concomitant increase in extracellular potassium. In the concentrations used, none of the cations, alone or in combination with colchicine, had any effect on total hormone production, the uptake of 3H-amino acids by the cells, or total cell protein synthesis. We conclude that depolarizing concentrations of extracellular potassium have a PRL- and GH-releasing effect in these spontaneously secreting tumor cells and that the presence of extracellular calcium is necessary for this effect of potassium to take place. Moreover, the release of these hormones is at least in part dependent on a colchicine-sensitive step. (Endocrinology93: 793, 1973)