Objectives An increase in antibiotic resistance of Escherichia coli , the most common pathogen in urinary tract infections (UTIs), is encountered worldwide. Optimal treatment of UTIs will contribute substantially to limit antibiotic use and antimicrobial resistance. This study determined trends in antimicrobial resistance of uropathogenic E . coli , which can be of use to optimize UTI guidelines. Methods During 1998–2005, E . coli from urine samples of patients attending urology services were collected in three regions in The Netherlands: north-east (NE, n = 1084), west (W, n = 1064) and south (S, n = 1212). The antibiotic susceptibility was determined using microbroth dilution following CLSI guidelines. E . coli ATCC 35218 and ATCC 25922 were used as reference strains. Results Amoxicillin resistance remained stable over time (37% to 47%), but was higher in the south (44%) compared with the other regions (40%; P < 0.02). Resistance to piperacillin increased from 4% (1998) to 32% (2005; P < 0.001), and resistance to fluoroquinolones increased from 6% to 13% ( P < 0.01). Interregional differences were observed for resistance to piperacillin (NE 10%, W 12%, S 14%; P < 0.05) and to fluoroquinolones (NE 7%, W 13%, S 8%; P < 0.001). Trimethoprim ± sulfamethoxazole resistance remained stable (27% to 37%), as did that of nitrofurantoin (4% to 9%). The percentage of strains with multidrug resistance (resistance to three or more groups of antibiotics) for each region increased over time ( P < 0.05). Conclusions Antibiotic resistance was fairly constant over time for most agents tested, except for piperacillin and the fluoroquinolones. Regional differences were observed for several compounds. National and regional surveillance of antibiotic resistance is important to keep therapeutic guidelines up-to-date and adequate for the treatment of resistant microorganisms.