In this paper, the effects of tityustoxin and ouabain on the release and mobilization of ACh from cytoplasmic and vesicular stores of slices of cortex from the brain of the rat are described. Tityustoxin induced an increase of release of ACh which was time-dependent and this effect was accompanied by a concomitant decrease of the ACh, measured in cytoplasmic and crude vesicular fractions. Unlike tityustoxin, ouabain did not increase the release and synthesis of ACh during a 2 min period of stimulation but later, at 5, 10 and 15 min, ouabain increased the release and synthesis of ACh from incubated slices of cortex. After 10 min, ouabain caused a reduction of the content of ACh from cytoplasmic and crude vesicular fractions.