Innervated strips of longitudinal muscle of guinea pig ileum were incubated with 3H-choline in the presence of physostigmine, after which the efflux of tritiated metabolites was studied. Under resting conditions 3H-acetylcholine, 3H-choline and other tritiated metabolites were released into the incubation medium. Analysis of the kinetics of the release of the metabolites indicated that the release occurred from at least 2 different compartments. Under the unstimulated conditions used, between 72 and 78% of the radio-activity remained in a third, non-releasable compartment. The release of 3H-acetylcholine showed spontaneous fluctuations which were positively correlated to spontaneous variations of the isometric tension of the preparation. Electrical field stimulation at low frequency (0.5 Hz) consistently increased the release of 3H-acetylcholine. The release of 3H-choline was increased only slightly or not at all by the electrical stimulation. For studying stimulation-evoked release of 3H-acetylcholine, it is suggested that the measurement of the total quaternary amine release is of similar value to measurement of 3H-acetylcholine itself. This approach considerably simplified the method. During 5 consecutive stimulation periods of 150 pulses each, the increase in the release of total quaternary amines diminished with time. By taking into account the reduction of the stores of radioactivity in the tissue during the experiment, correction for the negative trend could be made. When the preparation was stimulated with between 30 and 3000 pulses a close correlation was found between the increase in the release of total quaternary amines and the number of pulses delivered. The volley output of the quaternary amines was not constant, however, but diminished by increasing stimulation frequency.