Sendai virus-induced hemolysis, a manifestation of virus-red cell fusion, is inhibited by exposure of the virus to 50 microM and higher concentrations of trifluoperazine. Trifluoperazine does not disrupt the virus, since trifluoperazine-treated virus with no hemolytic activity sediments slightly faster than untreated virus on sucrose density gradients and contains viral proteins in proportions characteristic of untreated virus. Trifluoperazine affects the fusion protein to a greater extent than the hemagglutinin, since trifluoperazine-treated virus with no hemolytic activity is as active or nearly as active in agglutinating red cells. The partition coefficient of trifluoperazine between the virus membrane and buffer is lower at 4 degrees C than, but the same at 37 degrees C, as that between the red cell membrane and buffer. Nevertheless, virus-independent red cell lysis and inactivation of virus-mediated hemolysis occur when the red cell and viral membranes, respectively, contain similar concentrations of trifluoperazine. Furthermore, 13-28% more trifluoperazine is necessary to achieve either effect at 4 degrees C or at 25 degrees C than at 37 degrees C. Changes in the surface activity of trifluoperazine do not explain these results, insofar as the critical micellar concentration of (0.75 mM) and maximal reduction in surface tension by (40 dyn/cm) trifluoperazine are the same at 25 degrees C and 37 degrees C. The fluorescence of viral tryptophan decreases by approx. 25% when viral hemolysis is inactivated by trifluoperazine, by trypsin treatment or by heating at 100 degrees C for 5 min.