Protein S-palmitoylation is a reversible posttranslational modification of proteins with palmitate or other long-chain fatty acids. In the last five 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 signaling inputs. A family of protein acyltransferases or PATs is responsible for modifying proteins with palmitate or other long-chain fatty acids on the cytoplasmic face of cellular membranes. The signature feature is a DHHC (Asp-His-His-Cys)-cysteine rich domain that is the catalytic center 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 the enzymes as targets for therapeutic intervention. Accordingly, elucidating the mechanism and regulation of the DHHC proteins is important for understanding how palmitoylation manifests in physiology and pathophysiology. Our recent work has focused on the enzymology of DHHC PATs, addressing the mechanism of palmitate transfer to substrate and the quaternary structure of the enzymes. DHHC proteins acylate themselves upon incubation with palmitoyl-CoA, a process termed autoacylation. Both autoacylation and transfer of palmitate to substrate are dependent upon the cysteine within the DHHC motif. It has been suggested that autoacylation of the enzyme represents a transient acyl-enzyme transfer intermediate. To directly test this hypothesis, we performed single turnover assays with DHHC2 and DHHC3 and demonstrated that a radiolabeled acyl group on the enzyme transferred to the protein substrate, consistent with a two-step ping-pong mechanism. Enzyme autoacylation and acyltransfer to substrate displayed the same acyl-CoA specificities, further supporting a two-step mechanism. Evidence suggests that mutation of the catalytic cysteine within the DHHC motif results in a protein that acts as a dominant negative (Fukata M. et al. (2004); Fang C. et al. (2006). However, the mechanism by which the mutation interferes with the function of the wild type protein is unknown. One hypothesis is that DHHC proteins function as oligomers and that mixed oligomers of wild type and mutant enzyme are inactive. Support for this argument comes from coimmunoprecipitation experiments that suggest that DHHC3 forms homomultimers and heteromultimers with DHHC7 (Fang C. et al. (2006). We sought to determine whether DHHC proteins oligomerize in cells and in vitro. Bioluminescence resonance energy transfer experiments revealed that DHHC2 or DHHC3 self-associate when expressed in HEK-293 cells. Purified DHHC3 resolved as a monomer and dimer on blue native polyacrylamide gels. In intact cells and in vitro, catalytically inactive DHHC proteins displayed a greater propensity to form dimers. BRET signals were higher for the catalytically inactive DHHC2 or DHHC3 than their wild type counterparts. DHHC3 BRET in cell membranes was decreased by the addition of palmitoyl-CoA, a treatment that results in autoacylation of the enzyme. Cross-linking of purified DHHC2 reversibly inhibited its activity in vitro. The correlation of enzyme activity with the monomeric species suggests that oligomerization may represent a mechanism to regulate enzyme activity.