Reversible protein acetylation is controlled by the opposing actions of protein lysine acetyltransferases and deacetylations. Recent developments on the structure and biochemical mechanisms of histone acetyltransferases (HATs) have provided new insight into catalysis and substrate selection. Diverse families of HATs appear to perform a conserved mechanism of acetyl transfer, where the lysine-containing substrate directly attacks enzyme-bound acetyl-CoA. The ability of HATs to form distinct multi-subunit complexes provides a means to regulate HAT activity by altering substrate specificity, targeting to specific loci, enhancing acetyltransferase activity, restricting access of non-target proteins, and coordinating the multiple enzyme activities of the complex. In the case of newly discovered Rtt109 HAT, association with distinct histone chaperones directs substrate selection between N-terminal lysines (H3K9, H3K23) and those (H3K56) within the histone fold domain. Moreover, the ability of some HATs to utilize longer chain acyl-CoA (i.e. propionyl-CoA) as alternative substrates suggests a potential direct link between the metabolic state of the cell and transcriptional regulation.