The coronavirus glycoprotein E2, which is responsible for virus attachment to cell receptors and virus-induced cell fusion, was purified by solubilization of virions with Triton X-114 and phase fractionation. Native E2 and tryptic subunits of the glycoprotein were separated by size-exclusion high-performance liquid chromatography (HPLC). Two distinct 90 kD E2 subunits, which had identical electrophoretic mobilities when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were separated by hydroxyapatite HPLC in the presence of sodium dodecyl sulfate.