Naturally occurring chloramphenicol resistance in bacteria is normally due to the presence of the antibiotic inactivating enzyme chloramphenicol acetyltransferase (CAT) which catalyzes the acetyl-S-CoA-dependent acetylation of chloramphenicol at the 3-hydroxyl group. The product 3-acetoxy chloramphenicol does not bind to bacterial ribosomes and is not an inhibitor of peptidyltransferase. The synthesis of CAT is constitutive in E. coli and other Gram-negative bacteria which harbor plasmids bearing the structural gene for the enzyme, whereas Gram-positive bacteria such as staphylococci and streptococci synthesize CAT only in the presence of chloramphenicol and related compounds, especially those with the same stereochemistry of the parent compound and which lack antibiotic activity and a site of acetylation (3-deoxychloramphenicol). Studies of the primary structures of CAT variants suggest a marked degree of heterogeneity but conservation of amino acid sequence at and near the putative active site. All CAT variants are tetramers composed in each case of identical polypeptide subunits consisting of approximately 220 amino acids. The catalytic mechanism does not appear to involve an acyl-enzyme intermediate although one or more cysteine residues are protected from thiol reeagents by substrates. A highly reactive histidine residue has been implicated in the catalytic mechanism.