The ability of 57 Spanish microbiology laboratories in detecting and reporting beta-lactam resistance phenotypes in Escherichia coli and Klebsiella pneumoniae was evaluated. Laboratories received 6 well-characterized isolates expressing the most widespread extended-spectrum beta-lactamases (ESBLs) in Spain (4 CTX-M type, 1 TEM type, and 1 SHV type), 3 isolates producing AmpC-type enzymes (2 plasmid mediated and 1 E. coli hyperproducing its chromosomal AmpC), and 3 quality control strains. Ninety-one percent of laboratories recognized all ESBL producers correctly, and therefore, low error rates were observed when testing cephalosporins and aztreonam. The highest error rates were observed with combinations of penicillin plus beta-lactamase inhibitor, although more than 60% of cases were due to the interpretation made by the microbiologists. Correct recognition of all AmpC beta-lactamase-producing strains occurred in only 47.4% of laboratories. These isolates were wrongly reported as ESBL producers and penicillinase hyperproducers in 7.6 % and 5.8% of cases, respectively. Detection of the AmpC-type phenotype by Spanish laboratories needs to be improved.