An investigation was made of the effects of the snake venom neurotoxin, notexin, on acetylcholine turnover in rat brain P2 fractions using a gas chromatographic mass spectrometric assay for acetylcholine and choline. In contrast to earlier reports, we found a stimulation of the uptake and acetylation of labeled choline by toxin-treated P2 fractions. More significantly, notexin inhibited the release of this newly synthesized transmitter. These effects were found to be dependent on the dose of the toxin and the time of exposure of the P2 fraction to notexin. Longer exposure to notexin or experiments involving resuspension of notexin-treated P2 fractions appeared to result in considerable lysis of the transmitter-containing particles. Thus, notexin may alter acetylcholine compartmentation in the nerve ending and thereby affect acetylcholine synthesis.