Prion diseases in humans and animals are invariably fatal. Prions are composed of a disease-causing isoform (PrP(Sc)) of the normal host prion protein (PrP(C)) and replicate by stimulating the conversion of PrP(C) into nascent PrP(Sc). We report here that tricyclic derivatives of acridine and phenothiazine exhibit half-maximal inhibition of PrP(Sc) formation at effective concentrations (EC(50)) between 0.3 microM and 3 microM in cultured cells chronically infected with prions. The EC(50) for chlorpromazine was 3 microM, whereas quinacrine was 10 times more potent. A variety of 9-substituted, acridine-based analogues of quinacrine were synthesized, which demonstrated variable antiprion potencies similar to those of chlorpromazine and emphasized the importance of the side chain in mediating the inhibition of PrP(Sc) formation. Thus, our studies show that tricyclic compounds with an aliphatic side chain at the middle ring moiety constitute a new class of antiprion reagents. Because quinacrine and chlorpromazine have been used in humans for many years as antimalarial and antipsychotic drugs, respectively, and are known to pass the blood-brain barrier, we suggest that they are immediate candidates for the treatment of Creutzfeldt-Jakob disease and other prion diseases.