The purpose of this investigation was to establish the existence of a calcium-independent mechanism for catecholamine secretion from the adrenal medulla. Concentration-effect curves were obtained using isolated perfused (5 ml/min; 25 degrees C) bovine adrenal medullae stimulated with acetaldehyde (1-64 mM). Catecholamine secretion was the same in presence and in absence of calcium in the perfusion medium and was not diminished by the addition of magnesium (5 X 10-minus 3 M) to the calcium-free medium. Stimulation of 45-Ca-labeled adrenals with 23 mM acetaldehyde evoked secretion of catecholamines but not 45-Ca. Furthermore, examination of the distribution of 45-Ca and catecholamines in subcellular fractions (cytosol, nuclei, microsomes, mitochondria and chromaffin granules) from acetaldehyde-stimulated and nonstimulated glands showed that acetaldehyde stimulation caused no mobilization of 45-Ca from any of the intracellular pools, but significantly decreased the catecholamine content of a chromaffin granule fraction with a concomitant increase of catecholamines in the cytosolic fraction. These results establish an adrenomedullary secretory mechanism independent of both extracellular and intracellular calcium and indicate that acetaldehyde evokes catecholamine secretion by this calcium-independent mechanism, releasing the intragranular hormones into the cytoplasm prior to their extrusion from the chromaffin cell.