Cultured chromaffin cells were preincubated with digitonin to deplete endogenous ATP from the cell cytoplasm. Catecholamine release from these digitonin-pretreated cells was then studied in the presence and absence of exogenous ATP to elucidate a possible involvement of the cytoplasmic ATP in the exocytotic process. The preincubation of the cells with digitonin in the ATP-free permeabilizing medium resulted in a marked decline of the releasing response to a calcium challenge. Furthermore, the declined activity of catecholamine release caused by digitonin pretreatment was restored by the presence of ATP, but not by other adenine nucleotides, and this recovery was observed in a manner dependent on the concentration of ATP. These findings, therefore, seem to indicate that a decrease in the releasing activity of the digitonin-pretreated cells may be due to the removal of endogenous ATP from the cytoplasmic space of the cells, thus suggesting that the cytoplasmic ATP may be involved in the exocytotic mechanism of catecholamine secretion.