Micro-methods for the measurement of water spaces and intracellular K + were applied to dispersed rat adrenal cortical cells in order to follow changes in intracellular K + after alteration of steroidogenesis in vitro. Using unpurified zona glomerulosa cells, both serotonin and high[K +] medium (8.4 mmol/l) increased corticosterone output but only the latter stimulus increased intracellular K +. Zona glomerulosa cells purified by unit gravity sedimentation showed no changes in intracellular K + with serotonin or angiotensin but both stimulated corticosterone output. Conversely, ouabain at 10 −5 mol/l depressed intracellular K + of glomerulosa cells but did not alter basal or serotonin stimulated corticosterone output. A higher dose of ouabain, 5 × 10 −4 mol/l, further depressed intracellular K + and inhibited steroidogenesis in both the glomerulosa and fasciculata cells. Glomerulosa and fasciculata cells showed the same pattern of changes in intracellular K + with all of the agents tested. A variety of situations were observed where changes in intracellular K + of glomerulosa cells did not parallel those in steroidogenesis. These measurements therefore do not support the hypothesis that intracellular K + is the sole mechanism regulating steroidogenesis in these experiments.