The growing prevalence of antibiotic resistance in numerous pathogenic bacteria is a major public health concern and urgently requires the development of new therapeutic approaches. Multidrug resistant species that remain sensitive to chloramphenicol (CAM) treatment have engendered renewed interest in using this drug as a modern day antimicrobial agent. High-level resistance to CAM commonly is mediated by chloramphenicol acetyltransferase (CAT) which catalyzes the acetylation of CAM and renders the drug inactive. Of the three main types (CAT, CAT and CAT), CAT is of broad clinical significance. Despite this importance, understanding of the catalytic mechanism of CAT largely is extrapolated from studies of CAT. Here, pentapeptide scanning mutagenesis was used to generate a library of random insertions in CAT to gain a better understanding of structure-function relationships in the enzyme. Pentapeptide insertions in secondary structure elements which contain residues that form part of the CAT active site abolished CAM resistance in Escherichia coli. Insertions in secondary structures that have key roles in protein folding and CAM binding led to a reduction in resistance. In contrast, insertions in loop regions between the major secondary structure features exerted modest, if any, effects on CAM resistance. The analysis pinpoints regions of CAT that may serve as targets for the design of novel inhibitors that prevent the spread of CAM-resistant pathogens thereby enabling the drug to be re-deployed as a broad range antimicrobial agent. Moreover, regions of CAT that are tolerant of insertions may be suitable for the construction of bifunctional enzymes in which peptides, mini-proteins or amino acid tags are introduced at the permissive sites.