The uptake of [3H[acetylcholine ([3H]AcCho) into cholinergic synaptic vesicle ghosts purified from Torpedo electric organ was studied at concentration of [3H]AcCho ranging from 0.1 to 10 mM. The accumulated [3H]AcCho can be released either by hypoosmotic buffer or by low levels of the detergent Triton X-100. Kinetic analysis of the initial rate of [3H]AcCho uptake reveals temperature-dependent saturation kinetics which are best fitted by high-affinity (KTh approximately 0.3 mM) and low-affinity (KT) approximately 10 mM) vesicular [3H]AcCho transport systems. Several lines of evidence suggest that [3H]AcCho transport is mediated by vesicle-associated transport systems and not by a contaminant of other subcellular moieties such as the plasma membrane choline transport system. (i) The specific activity of the [3H]AcCho transport systems is higher in the purest vesicular fraction than in the less-pure fractions. (ii) Ghosts prepared from isolated synaptosomes manifest only low levels of low-affinity [3H]AcCho transport and no high-affinity [3H]AcCho transport. (iii) The vesicular AcCho transport systems lack some of the typical characteristics of synaptosomal choline transport, such as Na+ activation. (iv) The ratio of uptakes of [3H]AcCho and [3H]choline (10 microM) is about 5-fold higher in the pure vesicles than in isolated synaptosomal membranes. Addition of Mg2+-ATP decreases the rate of vesicular [3H]AcCho uptake by about 50%. The simultaneous addition of NaHCO3 and Mg2+-ATP results in activation of [3H]AcCho uptake to about 125% (relative to control), which is a 2.5-fold enhancement relative to the rate observed with Mg2+-ATP. The present findings demonstrate the presence of novel vesicle-associated AcCho transport systems. Their physiological role in the life cycle of the cholinergic synaptic vesicle and nerve terminal are discussed.