The direct effect of alpha 2-autoreceptors was studied by measuring the effects of piperoxan, an alpha 2-autoreceptor antagonist, and clonidine, an agonist on catecholamine exocytosis, from single bovine chromaffin cells in culture. Catecholamine release was elicited by stimulation with 100 microM nicotine and was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. These electrodes allowed the number of exocytotic release events to be monitored and reported as total charge for release following a specific stimulus. Repeated stimulation with 100 microM nicotine showed that total release caused by the second exposure to nicotine was 32% of the first, and release caused by the third exposure to nicotine was 80% of the second. Total release of catecholamine increased significantly after application of 20 microM piperoxan relative to a control application of balanced salt solution. Application of 20 microM piperoxan alone did not cause release. After the cells were incubated in culture medium containing 20 microM clonidine, a significant decrease in nicotine-stimulated catecholamine release was observed. These results confirm that there are autoreceptors on chromaffin cells and, when relatively high levels of catecholamine are released, the catecholamine stimulates the alpha 2- autoreceptors, which inhibits subsequent release through a negative feedback mechanism. In addition to piperoxan, the sympathomimetic drug amphetamine also increases quantal release after application of nicotine. Amphetamine increases the extracellular concentration of catecholamine, and these data appear to indicate that at least part of the pharmacology of amphetamine might involve blocking catecholamine autoreceptors.