A nationwide survey of extended-spectrum beta-lactamase (ESBL) production among Enterobacteriaceae, carried out in 2003, showed that CTX-M-type enzymes have achieved a sizeable prevalence among ESBL producers in Italy, mostly in Escherichia coli and, to a lesser extent, in Klebsiella pneumoniae. In this work, we report on the molecular epidemiology of the CTX-M-producing isolates from that survey and on the mechanisms of dissemination of these emerging resistance determinants. The CTX-M-producing isolates were detected in 10 of the 11 participating centers distributed across the Italian national territory, although at remarkably variable rates in different centers (1.2 to 49.5% of the ESBL producers). All CTX-M determinants were of group 1, with CTX-M-15 and CTX-M-1 being the most prevalent variants (60% and 35%, respectively) and CTX-M-32 carried by a minority (5%) of isolates. Each variant was detected both in E. coli and in K. pneumoniae. Genotyping of the CTX-M-producing isolates by random amplification of polymorphic DNA revealed a notable diversity, especially among those producing CTX-M-1, while clonal expansion was evident with some CTX-M-15-producing strains. Mating experiments revealed a higher overall transferability of bla(CTX-M-1) and bla(CTX-M-32) than of bla(CTX-M-15). Coresistance to quinolones and aminoglycosides was overall higher with the CTX-M-15-producing isolates. The present results indicate that CTX-M-producing strains are now widespread across the Italian territory and underscore the emerging role of these ESBL determinants in the European setting. They also reveal notable differences in the dissemination mechanisms of genes encoding different CTX-M variants of the same lineage.