AbstractElectrical field stimulation was applied to the rabbit lacrimal gland during incubationin vitrowith3H‐choline, after which the efflux of tritiated metabolites was studied. In the presence of physostigmine3H‐acetylcholine was released into the incubation medium when the gland was stimulated electrically. The identity of the3H‐acetylcholine synthesized by the preparation was established by its identical ion exchange chromatographic properties to native acetylcholine. The tissue contents of3H‐acetylcholine and3H‐choline were 14.4 and 3.6%, respectively, of the total activities found in the gland. This study demonstrates that the rabbit lacrimal gland is innervated cholinergically. It is suggested that the method developed might be of value for studying effects of drugs on cholinergic neurotransmission in t