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Abstract: Numerous proteins that are involved in cell signaling and viral replication require post‐translational modification by palmitoylation to function properly. The molecular details by which this palmitoyl modification affects protein function remain poorly understood. To facilitate in vitro biochemical and structural studies of the role of palmitoylation on protein function, a method was developed for alkylating peptides with saturated C[sub 16] groups at cysteine residues and demonstrated using peptides derived from the palmitoylated region of Sindbis virus E2 glycoprotein. The synthetic approach takes advantage of disulfide chemistry to specifically modify only the cysteine residues within peptides and covalently links C[sub 16] groups via disulfide bridges using a new thioalkylating reagent, hexyldexyl‐dithiopyridine. The chemistry presented here takes place in solution under mild conditions without the need for protection of the peptide functional groups. A method for purifying these modified peptides is also described. This protocol can be of general use to investigators studying the role of palmitoylation in biological systems. [ABSTRACT FROM AUTHOR]