To investigate the correlation between mutations in promoter, attenuator, and the AmpC enzyme overproduction in Escherichia coli. ampC Promoters from 4 Escherichia coli clinical isolates were cloned upstream to the chloramphenicol acetyltransferase (CAT) gene in pCAT3 reporter plasmid. Promoter strengths were measured by chloramphenicol MIC and gene sequencing was done on the cloned ampC promoter and attenuator. The strength of promoters from AmpC hyperproducers were 8-to 64-fold higher than those from a low-level AmpC producers. In one of the high-strength promoters, the mutations were located at positions -32, +22, +26, +32 (attenuator), -76, and +79. In another promoter, the mutations were located at positions -88, -82, -18, -1, and +58. In the third promoter, mutations were found at positions -1, +58, -80, -73, -28, and +82. Mutations in Escherichia coli promoter and attenuator sequences promoted Chloramphenicol MICs, which may be the primary causal mechanism for resistance to beta-lactams antibiotics.