Cefditoren is a third-generation orally administered cephalosporin with a broad spectrum of activity against Gram-positive and Gram-negative bacterial species. After an oral 400-mg single dose, the mean concentrations in urine are 186.5 mg/L at 2 to 4 h and 12.7 mg/L at 8 to 12 h, and it is a potential drug to be used in the treatment of urinary tract infection (UTI). We performed a multicenter nationwide study in Spain in order to determine the in vitro activity of cefditoren and other comparative agents against Enterobacteriaceae causing community-acquired uncomplicated UTI in women. From June 2008 to March 2009, 89 institutions participated in the study. A total of 2152 Enterobacteriaceae were collected and sent to a coordinating laboratory where identification and antimicrobial susceptibility testing was performed against 20 antimicrobials using an automated microdilution method (MicroScan; Siemens, Sacramento, CA). Cefditoren MICs were determined by the broth microdilution method (Clinical and Laboratory Standards Institute guidelines) using the same inoculum. Microorganisms isolated were Escherichia coli (81.8%), Klebsiella pneumoniae (7.9%), Proteus mirabilis (5.2%), and others (5.1%). A total of 51 isolates (2.4%) were extended-spectrum beta-lactamase (ESBL) producers, 3 (0.1%) produced plasmidic AmpC enzymes, and 64 (2.9%) produced chromosomal AmpC. The MIC(50)/MIC(90) (mg/L) of cefditoren against all isolates was 0.12/0.5. Cefditoren inhibited 96.5% of isolates at 1 mg/L and was uniformly active against all isolates with the exception of strains producing ESBLs or AmpC enzymes. The MIC(50)/MIC(90) of other antimicrobials were ampicillin (AMP) >16/>16, amoxicillin/clavulanic acid (A/C) <or=8/4/16/8, cefuroxime (FUR) <or=4/8, cefotaxime <or=1/<or=1, ciprofloxacin (CIP) <or=0.12/>2, trimethoprim/sulfamethoxazole (SxT) <or=2/38/>4/76, and fosfomycin (FOS) <or=16/<or=16. The respective percentages of resistance were 61%, 17.2%, 5.5%, 2.3%, 20.2%, 27.4%, and 4.8%. The activity of cefditoren against Enterobacteriaceae producing community-acquired uncomplicated UTI in women was superior to that of AMP, A/C, FUR, CIP, and SxT and similar to that of FOS.