A class of ampC beta-lactamase-hyperproducing mutants of Escherichia coli were shown to have the insertion element IS2 inserted into the ampC promoter. The insertion of IS2 in orientation II created a novel promoter in which the -35 region and the 17 bp long spacing sequence between the two consensus sequences are present in IS2 DNA, whereas the -10 region from the original ampC promoter is retained. In vitro transcription revealed that the transcription initiation site in the ampC::IS2 mutants was identical with that of ampC wild-type promoter. The novel promoter exhibited a 20-fold increase in promoter strength relative to the original ampC promoter, presumably due to the increase in the spacing sequence from 16 to 17 bp. The evolution of transposable elements and of control elements such as promoters are discussed on the basis of the findings described herein.