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The spike protein S of coronaviruses contains a highly conserved cytoplasmic cysteine-rich motif adjacent to the transmembrane region. This motif is palmitoylated in the Betacoronaviruses MHV and SARS-CoV. Here, we demonstrate by metabolic labeling with [(3)H]-palmitic acid that the S protein of transmissible gastroenteritis coronavirus (TGEV), an Alphacoronavirus, is palmitoylated as well. This is relevant for TGEV replication as virus growth was compromised by the general palmitoylation inhibitor 2-bromopalmitate. Mutation of individual cysteine clusters in the cysteine-rich motif of S revealed that all cysteines must be replaced to abolish acylation and incorporation of S into virus-like particles (VLP). Conversely, the interaction of S with the M protein, essential for VLP incorporation of S, was not impaired by lack of palmitoylation. Thus, palmitoylation of the S protein of Alphacoronaviruses is dispensable for S-M interaction, but required for the generation of progeny virions.