A number of fatty acids were found to inhibit Sendai virus-induced hemolysis. cis-Unsaturated fatty acids such as oleate, as well as the methyl-branched isostearate, completely inhibited viral hemolysis at concentrations as low as 5-10 micrograms/ml, whereas the saturated, normal acids such as palmitate and stearate were comparably inhibitory only at 2-5 times those concentrations. trans-Unsaturated acids, as well as several other amphiphilic compounds, were either not or only weakly inhibitory. In contrast to their disparate effects on viral hemolysis, cis- and trans-unsaturated acids lysed erythrocytes in the same concentration range, which is several times higher than that at which the former compounds inhibited viral hemolysis. The mechanism of inhibition of viral hemolysis by isostearate involves the inactivation of viral hemolytic activity per se, since isostearate neither inhibited viral hemagglutination nor rendered erythrocytes significantly less susceptible to hemolysis. Furthermore, the concentration dependence of hemolysis inhibition by isostearate was biphasic, increasing sharply at the critical micelle concentration from a linear relationship below that concentration. Finally, an inhibitory concentration of isostearate was well below that at which amphiphiles dissolved membranes and did not dissolve Sendai virus, as shown by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was concluded that low concentrations of fatty acids--particularly cis-unsaturated or fluid-phase types--could block the fusion, as opposed to agglutination, step of viral hemolysis by perturbing hydrophobic regions of the Sendai virus membrane.