The objective of this study was to determine the subcellular origin of cholinergic transmitter released spontaneously from mouse forebrain minces. To accomplish this objective, minces were pretreated in ionic media and then loaded with [14C]homocholine, an analog of choline, to form the false transmitter [14C]acetylhomocholine [( 14C]AHCh). The ratio of the false transmitter [14C]AHCh to the true transmitter ACh was then used as an index of cholinergic transmitter contents for both the cytoplasmic (S3) and vesicle-bound (P3) fractions. Three different pretreatment procedures were used to cause the following changes in S3 and P3 false to true transmitter ratios prior to spontaneous release: 1) a small increase in the S3 ratio of [14C]AHCh to acetylcholine (ACh) and a large increase in the P3 ratio of [14C] AHCh to ACh; 2) a decrease in the S3 ratio of [14C]AHCh to ACh and an increase in the P3 ratio of [14C]AHCh to ACh; 3) an increase in the P3 ratio of [14C]AHCh to ACh without affecting the S3 ratio of [14C]AHCh to ACh. The influence of each pretreatment on these subcellular ratios was then compared with its influence on the spontaneous release ratio of [14C]AHCh to ACh. In all 3 instances, the influence of pretreatment on the ratio of spontaneously released false and true cholinergic transmitters from minces coincided with the effect of pretreatment on the pre-release ratio of false to true transmitter in the S3 fraction. These results suggest that much of the cholinergic transmitter which is spontaneously released from mouse forebrain occurs from the cytroplasmic fraction.