Influenza viruses encoding hemagglutinin (HA) and neuraminidase (NA) glycoproteins with deletions in one or both cytoplasmic tails (HAt− or NAt−) have a reduced association with detergent-insoluble glycolipids (DIGs). Mutations which eliminated various combinations of the three palmitoylation sites in HA exhibited reduced amounts of DIG-associated HA in virus-infected cells. The influenza virus matrix (M₁) protein was also found to be associated with DIGs, but this association was decreased in cells infected with HAt− or NAt− virus. Regardless of the amount of DIG-associated protein, the HA and NA glycoproteins were targeted primarily to the apical surface of virus-infected, polarized cells. The uncoupling of DIG association and apical transport was augmented by the observation that the influenza A virus M₂ protein as well as the influenza C virus HA-esterase-fusion glycoprotein were not associated with DIGs but were apically targeted. The reduced DIG association of HAt− and NAt− is an intrinsic property of the glycoproteins, as similar reductions in DIG association were observed when the proteins were expressed from cDNA. Examination of purified virions indicated reduced amounts of DIG-associated lipids in the envelope of HAt− and NAt− viruses. The data indicate that deletion of both the HA and NA cytoplasmic tails results in reduced DIG association and changes in both virus polypeptide and lipid composition.