Six ampicillin-resistant clinical isolates of Escherichia coli that hyperproduced the chromosomal ampC beta-lactamase were studied. By DNA sequence analysis, we found that five of them were identical over an entire 449-base-pair sequence and carried a novel strong ampC promoter [Olsson, O., Bergström, S. & Normark, S. (1982) EMBO J. 1, 1411-1416]. Except for one base pair this sequence was identical to that of a low beta-lactamase-producing clinical isolate of Shigella sonnei. Spontaneous one-step mutants of S. sonnei that overproduced the ampC beta-lactamase by 45-fold were characterized and found to be mutated at the single base that distinguishes S. sonnei from the five E. coli hyperproducers. The most likely explanation for this result is that chromosomal DNA was transferred in vivo from Shigella to E. coli across the species barrier.