The mechanism by which tacrine increases the content and synthesis of acetylcholine (ACh) in cerebrocortical prisms exposed to an irreversible inhibitor of cholinesterases and incubated under resting conditions (Dolezal and Tucek, 1991) is not known. As found in the present experiments, this effect of tacrine is only apparent if its application had been preceded by a period of preincubation, but the preincubation is ineffective if it occurs in the presence of hemicholinium-3. Apparently, choline or a choline-containing compound accumulates in the slices during the preincubation and is then utilized for the enhanced synthesis of ACh in the presence of tacrine. Tacrine did not induce a decrease in the amount of radiolabel that had been incorporated from choline into acid-insoluble compounds, which suggests that the choline which is used for the synthesis of additional ACh does not originate from choline lipids. However, tacrine was found to diminish the efflux of choline from prisms which had been preincubated with an increased concentration of choline in the medium, and from prisms incubated in the presence of hemicholinium-3. It also diminished the efflux of radioactive choline that had accumulated in the prisms during preincubation with a very low concentration of tacrine, when the prisms were subsequently incubated with 4-aminopyridine. It is proposed that the potency of tacrine to increase the content and synthesis of ACh in cerebrocortical prisms whose cholinesterases had been inhibited is due to its ability to diminish the efflux of endogenous choline from the nerve terminals.