The dynamics of catecholamine storage were studied in highly purified, small synaptic vesicles from rat brain both during active uptake or after inhibiting uptake with reserpine, tetrabenazine, or removal of external dopamine. To assess turnover during active uptake, synaptic vesicles were allowed to accumulate [3H]dopamine ([3H]DA) for approximately 10 min and then diluted 20-fold into a solution containing unlabeled DA under conditions such that active uptake could continue. After dilution, [3H]DA was lost with single exponential kinetics at a half-time of approximately 4 min at 30 degrees C in 8 mM Cl- medium, in which both voltage and H+ gradients are present in the vesicles. In 90 mM Cl- medium, in which high H+ and Cl- gradients but no voltage gradient are present, [3H]DA escaped at a half-time of approximately 7 min. In both high and low Cl- media, approximately 40% of [3H]DA efflux was blocked by reserpine or tetrabenazine. The residual efflux also followed first-order kinetics. These results indicate that two efflux pathways were present, one dependent on DA uptake (and thus on the presence of external DA) and the other independent of uptake, and that both pathways function regardless of the type of electrochemical H+ gradient in the vesicles. The presence of both uptake-dependent and -independent efflux was observed in experiments using DA-free medium, instead of uptake inhibitors, to prevent uptake. Uptake-independent efflux showed molecular selectivity for catecholamines; [14C]DA was lost about three times faster than [3H]norepinephrine after adding tetrabenazine directly (without dilution) to vesicles that had taken up comparable amounts of each amine. In addition, the first-order rate constant for uptake-independent efflux showed little change over a 60-fold range of internal DA concentrations, which suggests that this pathway had a high transport capacity. All efflux was blocked at 0 degrees C, suggesting that efflux did not occur through a large pore. There was little or no change in the proton gradient in synaptic vesicles, monitored by [14C]methylamine equilibration, during the experimental manipulations used here. Thus, the driving force for catecholamine uptake remained approximately constant. The physiological role of uptake-independent efflux could be to allow the monoamine content of synaptic vesicles to be regulated over a time range of minutes and, thereby, control the amount released by exocytosis. These results imply that catecholamines turn over with a half-time of minutes during active uptake by brain synaptic vesicles in vitro.