We have studied the binding of the Semliki Forest virus and its isolated spike glycoproteins, in the form of water-soluble octameric complexes, to various cells at 5 degrees C. The number of viruses bound per cell increased strongly with increasing free concentrations of virus up to about 0.2 nM. At higher concentrations smaller increases in binding were observed but saturation was not achieved. The number of viruses bound at a given free concentration was widely different for different cells. For some cells the binding of the virus was maximal at pH 6.8 with little decrease at lower pH, for other cells it was maximal around pH 6.0. The spike protein complexes were used at 100 times higher molar concentrations than the virus. The binding increased strongly with increasing free concentrations up to about 50 nM and saturation was obtained at higher concentrations. Up to 1.3 X 10(6) spike protein complexes could be bound per cell but great variation could be seen between different cell types. For all cells maximal binding was found below pH 6.0. Together with earlier observations, our results suggest that the virus can bind to a cell by two different modes. Around neutral pH the virus binds to specific glycoproteins and at low pH unspecifically to the lipids of the plasma membrane. The possible physiological roles of these two types of binding are discussed.