The ligand binding relationship between the acetylcholine transporter (AcChT) and the vesamicol receptor (VR) and the kinetics of active transport were studied in synaptic vesicles purified from the Torpedo electric organ using analogues of AcCh and vesamicol. Methoxyvesamicol, which should exhibit better equilibration properties for kinetics measurements than the more potent parent, inhibits active transport in a nonlinear noncompetitive manner. AcCh analogues competitively inhibit binding of [3H]vesamicol with higher affinity in hyposmotically lysed vesicle ghosts than in intact vesicles, apparently due to removal of a competing internal, osmotically active factor. AcCh and actively transported analogues of AcCh that are up to 57% larger in van der Waals volume exhibit up to a 200-fold ratio for the dissociation constant measured by inhibition of vesamicol binding to ghosts (KIAg) compared to the Michaelis constant for transport (KM) or the IC50 value for inhibition of [3H]AcCh active transport. In contrast, two AcCh analogues that are about 120% larger and that almost surely are not transported exhibit a KIAg/IC50 ratio of about 1. The data demonstrate that the vesamicol family of compounds binds to an allosteric site in the AcChT. Initiation of active transport has no apparent effect on the affinities of vesamicol and AcCh analogues, which suggests that most of the AcChT-VR in purified vesicles is transport incompetent. Vesicle ghosts actively transport [3H]AcCh nearly as well as intact vesicles, which suggests that internal factor does not affect transport-competent AcChT-VR. A kinetics model is proposed that predicts that AcCh analogues exhibiting a KIAg/IC50 ratio significantly greater than 1 are actively transported. Some of the microscopic constants in the model are estimated. The AcChT binds AcCh very weakly with a dissociation constant of about 20-50 mM, but it transports substrates rapidly in a process exhibiting remarkably little selectivity for the detailed shape and volume of the transported ion.