Ten phenothiazine derivatives, substituted in the 2- and 10-positions, were tested as inhibitors of a conditioned avoidance response in rats. The intraperitoneal activities, in decreasing order were: fluphenazine = SQ 10,037 > SQ 4924 = SQ 10,036 > perphenazine > trifluoperazine > thipropazate > triflupromazine > prochlorperazine > chlorpromazine. A CF 3 group in position 2 in place of Cl increased activity, and incorporating the N of the side chain in a piperazine ring increased activity still more. A direct comparative study in conditioned rats showed fluphenazine and acetyl-fluphenazine (SQ 4924) to be equally potent and 7 to 9 times as active as triflupromazine. When duration of action was included in the evaluation of activity, fluphenazine and acetylfluphenazine were 25 times as active as triflupromazine. In operant behavior studies in rats, fluphenazine was somewhat slower acting than triflupromazine but was more potent and longer acting. Fluphenazine was 6.5 times as potent as triflupromazine as a “tranquilizer” in hostile rhesus monkeys. The effects of fluphenazine and triflupromazine on behavior and body temperature of dogs were not significantly different. Both drugs reduced spontaneous motor activity, caused sedation and ataxia, and decreased rectal temperature. Cumulative, intravenous doses of fluphenazine or triflupromazine caused slight to moderate hypotension in anesthetized and unanesthetized dogs. Cumulative, intravenous administration of large doses of fluphenazine or triflupromazine to cats caused progressive bradycardia, marked transient hypotension and cardiac arrest. No changes in EEG were noted. At the peak of action, fluphenazine was approximately 4.5 times as potent as triflupromazine as an inhibitor of an elicited head-twitch response in mice. Fluphenazine and triflupromazine caused motor deficits in mice and were equally toxic. In general, fluphenazine (or its acetyl ester) appears to be a more potent and longer-acting tranquilizing agent than any other compound tested and this activity is unattended by corresponding increases in toxic or undesirable autonomic side actions.