The role of phospholipids in vaccinia virus was investigated by substituting viral lipids with specific phospholipids. Treatment of virus with sodium dodecyl sulfate, sodium deoxycholate, or Nonidet-P40 (NP-40) resulted in almost complete removal of viral lipid and led to inactivation of the virus. The inactivation induced by the former two was irreversible, but NP-40-treated virus was reactivated upon reassociation with phospholipids. Individual phospholipids, including phosphatidylserine (PS), phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, lysolecithin, sphingomyelin, and acyl bis(monoacylglycero)phosphate (ABMP), were tested for ability to reactivate NP-40-treated virus. Reactivation was induced only by PS. The infectivity of virus that had been treated with NP-40 and then with PS was unstable; the reactivated virus was inactivated within a short period. It was also very sensitive to trypsin. Treatment of NP-40-treated virus with mixtures of PS and ABMP yielded virus that was more resistant to spontaneous and trypsin-induced inactivation. Thus, PS appears to be an essential for infectivity and ABMP appears to play a supplementary role for maintenance of infectivity, perhaps by protecting against inactivating factors.