ABSTRACT Shotgun cloning experiments with restriction enzyme-digested genomic DNA from Morganella morganii 1, which expresses high levels of cephalosporinase, into the pBKCMV cloning vector gave a recombinant plasmid, pPON-1, which encoded four entire genes: ampC , ampR , an hybF family gene, and orf-1 of unknown function. The deduced AmpC β-lactamase of pI 7.6 shared structural and functional homologies with AmpC from Citrobacter freundii , Escherichia coli , Yersinia enterocolitica , Enterobacter cloacae , and Serratia marcescens . The overlapping promoter organization of ampC and ampR , although much shorter in M. morganii than in the other enterobacterial species, suggested similar AmpR regulatory properties. The MICs of β-lactams for E. coli MC4100 ( ampC mutant) harboring recombinant plasmid pACYC184 containing either ampC and ampR (pAC-1) or ampC (pAC-2) and induction experiments showed that the ampC gene of M. morganii 1 was repressed in the presence of ampR and was activated when a β-lactam inducer was added. Moreover, transformation of M. morganii 1 or of E. coli JRG582 (Δ ampDE ) harboring ampC and ampR with a recombinant plasmid containing ampD from E. cloacae resulted in a decrease in the β-lactam MICs and an inducible phenotype for M. morganii 1, thus underlining the role of an AmpD-like protein in the regulation of the M. morganii cephalosporinase. Fifteen other M. morganii clinical isolates with phenotypes of either low-level inducible cephalosporinase expression or high-level constitutive cephalosporinase expression harbored the same ampC-ampR organization, with the hybF and orf-1 genes surrounding them; the organization of these genes thus differed from those of ampC-ampR genes in C. freundii and E. cloacae , which are located downstream from the fumarate operon. Finally, an identical AmpC β-lactamase (DHA-1) was recently identified as being plasmid encoded in Salmonella enteritidis , and this is confirmatory evidence of a chromosomal origin of the plasmid-mediated cephalosporinases.