The anticholinesterase tacrine induces tremulous jaw movements in rats, and considerable evidence indicates that this response is dependent upon ventrolateral striatal mechanisms. Three experiments were conducted to study the relation between ventrolateral striatal acetylcholine and the production of tremulous jaw movements. In Experiment 1, intracranial microinjection of the acetylcholine synthesis inhibitor hemicholinium-3 into the ventrolateral neostriatum reduced tremulous jaw movements induced by 5.0 mg/kg tacrine. Microinjection of hemicholinium into a cortical site dorsal to striatum (Experiment 2) was without significant effect upon tacrine-induced tremulous jaw movements. In Experiment 3, rats were implanted with dialysis probes in the ventrolateral striatum to measure extracellular levels of acetylcholine during tacrine-induced jaw movements. Tacrine (2.5-5.0 mg/kg) increased both extracellular acetylcholine and tremulous jaw movements. The 5.0 mg/kg dose of tacrine produced a substantial increase in ventrolateral striatal acetylcholine levels (324% of baseline within 30 min). Across all tacrine-treated rats there was a significant linear correlation between tremulous jaw movements and acetylcholine levels (r = +0.56) during the first 30-min postinjection period. This correlation was largely due to the group that received 5.0 mg/kg tacrine; within this group, there was a very high correlation (r = +0.87) between tremulous jaw movements and acetylcholine levels in the first sample after injection. These data are consistent with the notion that tremulous jaw movements induced by tacrine are mediated by ventrolateral striatal acetylcholine. Moreover, these results suggest that dialysis methods could be used to monitor the relation between striatal acetylcholine and tremulous movements induced by a variety of different conditions.