Secretion of catecholamines from individual vesicles of bovine adrenal medullary cells was studied with amperometry in media of various osmolarities and compared with results obtained in isotonic physiological buffer (315 mosM). Hypotonic solutions caused an increase in the number of amperometric spikes evoked by brief exposure to 5 mM Ba2+. Under moderate hypertonic conditions (630 mosM), individual vesicular events were decreased in frequency, and lower amounts were secreted per event. Furthermore, the events were temporally broadened relative to those observed during release in isotonic conditions. At 970 mosM, exposure to 5 mM Ba2+ evoked even smaller secretory events that resemble the prespike feature that has been attributed to the initial opening of the fusion pore. The lack of large spikes is not due to failure of Ba2+ entry because fura-2 fluorescence reveals an increase in intracellular divalent ions. After exposure to Ba2+ in hypertonic solution, spikes could be induced with isotonic solution transiently directed onto the cell, but this process was not accompanied by a change in the concentration of intracellular divalent ions. Thus, this procedure provides an unique opportunity to temporally separate exocytotic secretion from entry of divalent ions.