The presence of 5 or 20 microM choline in the eserinized medium superfusing striatal slices enhanced the spontaneous release of acetylcholine (ACh) at both concentrations and, at 20 microM, the release of transmitter evoked by electrical field stimulation. Neither the electrical stimulation nor the addition of choline altered choline acetyltransferase activity. These results show that ACh release is dependent on the availability of extracellular choline. The rate of choline efflux was 7 times higher than the rate of ACh release, was not affected by stimulation, and was increased by 40% when hemicholinium-3 (HC-3), an inhibition of choline uptake, was present. The muscarinic antagonist atropine (1 microM) increased the evoked release of ACh into both the choline-free medium and that containing 20 microM choline. An adenosine receptor antagonist, 1,3-diethyl-8-phenyl xanthine (10 microM), failed to affect ACh release or the enhancement of release produced by atropine. In medium containing HC-3, stimulation of the slices elicited ACh release for the first 20 min of the 30 min stimulation period (15 Hz); thereafter, although stimulation was continued, the rate of release decreased to that associated with spontaneous release. Tissue ACh contents were not modified by the addition of choline or atropine to the medium, but were depressed by HC-3. Neither atropine nor HC-3 altered tissue choline content. The total amount of ACh + choline released during an experiment was 5-15 times higher than the decrease in tissue levels of these two compounds during the same period of time.(ABSTRACT TRUNCATED AT 250 WORDS)