Hemagglutinin (HA) of influenza virus is S-acylated with stearate at a transmembrane cysteine and with palmitate at two cytoplasmic cysteines. The amount of stearate varies from 35 (in avian strains) to 12% (in human strains), although the acylation region exhibits only minor or even no amino acid differences between HAs. To address whether matrix proteins and neuraminidase affect stearoylation of HA, we used mass spectrometry to analyze laboratory reassortants containing avian virus HA and the internal proteins from a human virus. Only minor fluctuations in the amount of stearate were observed, implying that other viral proteins do not affect acylation of HA.