Abstract : Incubation of a crude synaptosomal fraction from rat striatum with GBR 12783 at 37°C produced an inhibition of the specific uptake of [3H]dopamine that increased with time. The inhibition increased when GBR 12783 was present during preincubation and incubation (IC50 = 1.85 ± 0.1 nM) instead of incubation alone (IC50 = 25 ± 3.5 nM). Time-course studies of uptake inhibition demonstrated that a first collision transporter-inhibitor complex (TI) was formed immediately after addition of GBR 12783 so that the initial uptake velocity (Vo) decreased for increasing concentrations of inhibitor (Ki≥ 20 nM). TI slowly isomerized to a more stable complex TI* (K*i≤ 5 nM) with a value of t1/2 = 20-270 s. Fits of data to model 2 in which the steady-state uptake (VS) is set to zero were generally preferred, suggesting that formation of TI* could tend to irreversibility, as a consequence of a very low reverse isomerization. As expected, k, Vo, and VS tended to steady-state values in an asymptotic manner for high concentrations of GBR 12783. GBR 12783 at 2.5 nM produced a mixed inhibition of the uptake, with an increase in KM and a decrease in Vmax ; these effects were improved for 10 nM GBR 12783 and at 20°C. These results are discussed in relation to previous data concerning [3H]GBR 12783 binding. The present work gives the first experimental demonstration that dopamine uptake blockers can act according to a two-step mechanism of inhibition ; this is of great interest, because these inhibitors can oppose the effects of cocaine or amphetamine on the transporter according to a reaction that is partly nondependent on the concentration of the abused agent.