Few treatment options are available for cisplatin-refractory urothelial carcinoma. We evaluated the efficacy and safety of a new regimen composed of gemcitabine and ifosfamide as a second-line salvage chemotherapy for the disease. The gemcitabine and ifosfamide regimen consists of gemcitabine 800 mg/m/day intravenously for 30 min on days 1, 8, and 15; ifosfamide 1500 mg/m/day intravenously for 24 h on days 8-10; and mesna 800 mg intravenously bolus before ifosfamide and 1500 mg/m/day intravenously for 24 h on days 8-11. Cycles are repeated every 28 days. Between 1998 and 2005, 23 patients (median age 66) unresponsive to cisplatin-based chemotherapy (n=10) or who had tumor progression within 6 months of a previous response to cisplatin-based therapy (n=13) were enrolled. The median interval between the two chemotherapy regimens was 1.8 months (range 0.9-5.6). In total, 82 treatment cycles (median 3, range 1-8) were given. The overall response rate was 22% (95% confidence interval 5-39) with one complete response and four partial responses. Twenty-one patients succumbed to the disease. The median progression-free survival and overall survival were 3.5 and 4.8 months, respectively. Grade 3 or 4 leukopenia and thrombocytopenia occurred in 10 and eight patients, respectively. One, two and two patients complicated with grade 3 vomiting, diarrhea and stomatitis were present, respectively. No grade 3 or 4 neurotoxicity or nephrotoxicity was seen in these patients. The gemcitabine and ifosfamide regimen has an acceptable toxicity profile, but shows insufficient clinical activity in patients with cisplatin-refractory urothelial carcinoma to warrant further testing.