The main purpose of the study was to investigate the frequency of ESBL-producing E. coli and Klebsiella strains in the Greater Copenhagen area. Four collections of strains were investigated: A) 380 consecutive E. coli and Klebsiella isolates primarily from urine, B) 200 gentamicin-resistant E. coli and Klebsiella isolates primarily from urine, C) 210 consecutive E. coli isolates from blood cultures, and D) 68 cefuroxime-resistant E. coli and Klebsiella isolates primarily from urine. Only one strain per patient was included. Strains with a zone diameter for cefpodoxime <or=23 mm were tested by a phenotypic confirmatory test for ESBL production and all screening test-positive strains were examined with PCR and nucleotide sequencing in order to detect the following ESBL genes: ctx-m, shv, tem and oxa. Strains resistant to cefoxitin were further examined with cefotetan+/-boronic acid in order to detect AmpC. An ESBL gene was detected in 3/3 confirmatory test-positive isolates from collection A, in 14/17 from collection B, and in 41/48 from collection D. The distribution of isolates with the ESBL and/or AmpC enzymes was as follows: CTX-M (n=41), SHV (n=14), AmpC (n=9), CTX-M and AmpC (n=2), SHV and AmpC (n=1). In conclusion, the frequency of ESBL-producing E. coli and Klebsiella isolates was low in the Copenhagen area of Denmark (0.8 %). The most common ESBL genes found in our study were ctx-m and shv genes.