Background/Aims: Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) are urinary biomarkers of diagnostic relevance in a wide variety of acute and chronic kidney diseases. Their diagnostic sensitivity and specificity for kidney cancer are largely unknown and therefore the subject of this investigation. Methods: A prospective cohort study was performed to evaluate urine biomarkers for clear-cell and papillary subtypes of renal cancer (67 patients undergoing nephrectomy) and 55 control patients undergoing non-kidney surgery. Urinary KIM-1 and NGAL concentrations were determined by sensitive and specific ELISAs. Results: In renal cancer patients, median NGAL excretion was 0.52 (1st to 3rd quartiles: 0.28–0.82) ng/mg urinary creatinine (UCr) before nephrectomy compared to 0.15 (0.04–0.31) ng/mg UCr in controls (p < 0.001), and there was a modest decrease of 30% after nephrectomy (p < 0.008). NGAL was not correlated to tumor size (r = 0.19, p = 0.27) or stage. Before nephrectomy, KIM-1 excretion was 0.68 (0.40–1.12) ng/mg UCr compared to 0.03 (0.01–0.06) in controls (p < 0.001). There was a linear correlation between KIM-1 excretion before nephrectomy and tumor size (Spearman’s r = 0.66, p < 0.001), tumor stage, and a 50% decrease in median KIM-1 concentration 1 month following tumor excision (p < 0.01). Biomarker concentration ranges for renal cancer patients and controls overlapped substantially for NGAL but not KIM-1. Conclusion: NGAL is not a sensitive or specific urinary biomarker of kidney cancer. Although KIM-1 had diagnostic sensitivity for kidney cancer, it is well known to reflect many types of kidney injuries, thus limiting its specificity as a diagnostic biomarker for renal cancer.