To study the regulation of striatal acetylcholine (ACH) release, adult male rat striata were dissociated and incubated with 3H-choline to synthesize 3H-ACH. Fractional 3H-ACH efflux per min during continuous perifusion was: (1) tightly regulated; (2) dependent on calcium influx; (3) stimulated by 10 mM K+ and 1 mM glutamate; and (4) comparable to ACH release detected by HPLC. Thus, acutely dissociated striata exhibit calcium-sensitive, voltage-dependent secretion of 3H-ACH and direct receptor-mediated stimulation of release through the glutamate receptor family. This new approach toward cholinergic secretory physiology will help clarify complex striatal circuitry.