During the course of a molecular epidemiology study of mechanisms of antibiotic resistance in the area served by our hospital (516,000 inhabitants), we isolated the gene encoding CTX-M-14 beta-lactamase. Thirty clinical strains (27 Escherichia coli and 3 Klebsiella pneumoniae isolates) with a phenotype of extended-spectrum beta-lactamase were collected from January to October 2001 and studied for the presence of the CTX-M-14 beta- lactamase gene. By isoelectric point determination, PCR, and nucleotide sequencing, we detected the presence of this gene in 17 E. coli strains belonging to 15 different genotypes (REP-PCR) causing infections in 17 different patients. Epidemiological studies based on medical records did not suggest any relationship between the patients infected with these E. coli strains and, interestingly, 7 of 30 patients harboring strains with extended-spectrum beta-lactamases never had contact with the hospital environment before the clinical E. coli isolation. Conjugation experiments revealed that this gene was plasmid mediated in the 17 E. coli strains, and plasmid restriction fragment length polymorphisms showed 9 different patterns in the 17 E. coli strains. By PCR, the sequence of the tnpA transposase gene of the insert sequence ISEcp-1 was detected in all the plasmids harboring the CTX-M-14 gene. These results strongly suggest that plasmid dissemination between different E. coli strains in addition to a mobile element (transposon) around the beta-lactamase gene may be involved in the spreading of the CTX-M-14 gene. This study reinforces the hypothesis that the epidemiology of the prevalence of the beta-lactamase genes is changing and should alert the medical community to the increase in the emergence of the CTX-M beta-lactamases worldwide.