Exposure of influenza B virus-infected MDCK cells to chloroquine at the time of infection resulted in significant inhibition of infection. The appearance of input virus in the intracellular vesicles was not affected in the presence of the drug, but primary transcription of the virus genome did not occur. Chloroquine caused a rapid rise in the pH inside the lysosomes of MDCK cells, to 6.5 from the physiological pH 5.6. In contrast, exposure of infected cells incubated in acidic medium (pH 6.0) to chloroquine did not cause an increase in lysosomal pH and this low pH treatment during the chloroquine-sensitive phase was followed by virus production. Influenza B virus induced haemolysis of chick erythrocytes at low pH values (5.0 to 5.9) which was associated with cell-cell membrane fusion. It is likely that chloroquine prevents the uncoating of influenza B virus by increasing the lysosomal pH above the critical value required for inducing fusion between the virus envelope and the lysosomal membrane.