A total of 33 clinical isolates encoding TEM-3 (CTX-1) from four French hospitals were studied. The strains belonged to seven species, Klebsiella pneumoniae (n = 24), Escherichia coli (n = 3), Serratia marcescens (n = 2), Citrobacter freundii (n = 1), Enterobacter aerogenes (n = 1), Enterobacter cloacae (n = 1), and Klebsiella oxytoca (n = 1). All the strains harbored an Inc7 or M self-transferable plasmid with a size of approximately 85 kilobases. The plasmids had closely related EcoRI, HincII, HindIII, and PvuII restriction endonuclease-generated patterns and conferred resistance to all beta-lactams, except cephamycins and imipenem; to tetracycline, because of the presence of the genes blatem-3 and tetC, respectively, as determined by hybridization with specific probes; and to sulfonamide. Depending on the presence or absence and level of expression of the genes aacA4, aadA, and dfrI and of insertion element IS15, four types of plasmids could be distinguished. Plasmid pCFF04, the prototype plasmid encoding TEM-3, was widespread and appeared, by Southern hybridization, as the progenitor of the other types of replicons. The plasmid epidemic responsible for dissemination of TEM-3 in clinical isolates of members of the family Enterobacteriaceae may have originated in S. marcescens since pCFF04 was first detected in this species.