Tremorine (l,4-dipyrrolidino-2-butyne), a tremorigenic agent used for screening antiparkinsonian drugs, was activated in vivo and in vitro by incubation with liver. The activated product could be distinguished from Tremorine by its different biological properties. Activated Tremorine elicits tremors and parasympathetic-like stimulation in mice immediately after an intravenous dose of 3 mg/kg while Tremorine requires approximately 15 min. The cat latent period, after an intraperitoneal injection, was reduced from 15-30 min. to 5 min. Diethylaminodiphenyl-propyl acetate (SKF-525A) an inhibitor of liver microsomal activity, blocks the effects of Tremorine but does not alter the response of activated Tremorine. Isolated rabbit gut was unaffected by Tremorine (3.5 x 10-5 M) while activated Tremorine in equivalent concentration markedly stimulated the gut. Tremorine (5 mg/kg) had no consistent immediate effect on the blood pressure of anesthetized dogs while doses as small as 1.0 ug/kg of activated product produced an immediate bradycardia and a significant fall in blood pressure. Material having the biological properties of activated Tremorine can be extracted from an alkalinized solution by chloroform. Such material was also found in the urine of rats and mice given Tremorine and in solutions of Tremorine incubated with hamster liver slices and with hamster and rat liver homogenates.