Co-trimoxazole resistance in Escherichia coli isolates from urinary tract infections (UTI) was assessed in 382 strains from 1995 and 510 strains from 2005. The strains were collected from five microbiology laboratories in Ankara, Turkey. Documentation on patient gender, age and outpatient/inpatient status was collected in 2005, but not in 1995. The resistance percentages were 75.1% in 1995 and 55.5% in 2005. This reduction in resistance percentage was statistically significant, overall in all except two of the participating laboratories. The resistance percentage in 2005 was 61.1% for children (n = 208) and 51.2% for adults (n = 258), 53.7% for females (n = 380) and 60.8% for males (n = 130), and 55.3% for outpatients (n = 400) and 56.4% for inpatients (n = 110). The reduction in resistance is believed to be a consequence of reduced usage. Although decreased, the level of co-trimoxazole resistance remains high, and continued avoidance of its use for empiric treatment of UTI in Turkey appears to be an appropriate strategy.