Treatment of cultured rat pituitary GH3 cells with 50 mM KC1 in growth medium released 33% of cell PRL and 18% of cell GH with a half-time of 5 min. Hormone in the culture medium was increased 2- to 4- fold over unstimulated levels. The response required calcium; barium and strontium, but not magnesium, could substitute for calcium. Low temperature completely inhibited hormone release, which was also reduced significantly by inhibitors of energy metabolism and by nitrogen. This acute response was similar in ionic requirements, hormones released, and time course to the acute effect of TRH. Like potassium stimulation, TRH resulted in acute release of both PRL and GH. This contrasts with the finding that chronic TRH treatment reduced GH synthesis in GH3 cells. After a 10-min preincubation with potassium, subsequent short incubations with potassium released little hormone unless the cells were allowed to recover by incubation in normal medium for at least 2 h. This acutely releasable hormone pool seems to be located in a membrane-bound subcellular fraction, since GH3 cells did not discharge the cytoplasmic marker enzyme, lactate dehydrogenase, during potassium-stimulated hormone release.