The two glycoproteins that form the external spikes of the alphaviruses are type 1 membrane proteins whose transmembrane domains of hydrophobic amino acids are close to the carboxyl termini of the polypeptides and anchor the proteins in the lipid bilayer. Most of the members of the alphavirus genus contain within this transmembrane sequence one or more highly conserved cysteines, which are positioned close to the cytoplasmic face of the lipid bilayer. Cysteines in the cytoplasmic domains of the alphavirus glycoproteins and other enveloped viruses have been shown to be modified by palmitylation. To determine whether the transmembranal cysteines in Sindbis virus also were palmitylated, we used site-directed mutation to change the single transmembranal cysteine in the E1 glycoprotein and two of the transmembranal cysteines in the E2 glycoprotein to alanines. Transfection of RNA transcribed from the differently mutated Sindbis virus cDNAs led to production of infectious virus. Cells infected with the mutant virions and labeled with [³H]-palmitic acid showed that the E1 mutant no longer contained fatty acid in the E1 glycoprotein and that the extent of palmitylation was reduced about twofold in the E2 glycoprotein of virions containing the E2 mutations. At early times postinfection, the mutants grew slightly slower than the wild type in cultures of chicken embryo fibroblasts and secreted about half the amount of virus particles as wild type, but little difference was found at later time points. A triple mutant containing both the E1 and E2 mutations formed virions deficient in palmitylation of both glycoproteins, and this mutant had growth properties that were similar to those of the independent E1 and E2 mutants. Virions with the mutated glycoproteins that were deficient in fatty acid were more susceptible than the wild-type virions to inactivation by the detergent Triton X-100.