Palmitoylation is a post-translational modification that occurs on selected cysteines of many proteins. Since a high proportion of basic and hydrophobic residues is often found near the palmitoylated cysteine, the role of these residues in the selection of specific palmitoylation sites was assessed. Short peptides derived from the β2-adrenergic receptor sequence, modified to present different proportions of basic, acidic and hydrophobic residues, were tested in an in vitro S-acylation assay. Basic residues proved to be essential, whereas hydrophobic residues greatly enhanced S-acylation and acidic residues inhibited it. Taken together, these results show that short peptides contain the required molecular determinants leading to selective S-acylation. Whether or not these sequence characteristics also contribute to the selectivity of palmitoylation in vivo will need to be further investigated.