Protein S-palmitoylation is a reversible post-translational modification of proteins with fatty acids. In the last 5 years, improved proteomic methods have increased the number of proteins identified as substrates for palmitoylation from tens to hundreds. Palmitoylation regulates protein membrane interactions, activity, trafficking and stability and can be constitutive or regulated by signalling inputs. A family of PATs (protein acyltransferases) is responsible for modifying proteins with palmitate or other long-chain fatty acids on the cytoplasmic face of cellular membranes. PATs share a signature DHHC (Asp-His-His-Cys) cysteine-rich domain that is the catalytic centre of the enzyme. The biomedical importance of members of this family is underscored by their association with intellectual disability, Huntington's disease and cancer in humans, and raises the possibility of DHHC PATs as targets for therapeutic intervention. In the present paper, we discuss recent progress in understanding enzyme mechanism, regulation and substrate specificity.