The fusion of influenza virus with cultured cells has been investigated. The virus was labelled with the fluorescent probe octadecyl rhodamine B and fusion was monitored as fluorescence dequenching due to dilution of the probe from the viral into a cellular target membrane. Fusion with the plasma membrane does not occur, unless the extracellular pH is temporarily lowered. At neutral pH fusion occurs only after a lag phase of 10-15 min, the time required for virus internalization, and the reaction is inhibited by NH4Cl, indicating that it takes place in an intracellular acidic compartment, most likely the endosome. This suggests that influenza virus infects cells via the endocytic pathway.