Background: Use of aprotinin has been associated with acute kidney injury after cardiac surgery. Neutrophil gelatinase-associated lipocalin (NGAL) is a novel, very sensitive marker for renal injury. Urinary NGAL may be able to detect renal injury caused by aprotinin. This study determined if the use of aprotinin is associated with an increased incidence of acute kidney injury and increased levels of urinary NGAL. Methods: In this prospective, observational study 369 patients undergoing cardiac surgery were enrolled. 205 patients received aprotinin and 164 received epsilon amino-caproic acid intraoperatively. Urinary NGAL was measured before and immediately after cardiac surgery and 3, 18 and 24 h later. The association of aprotinin use with the incidence of acute kidney injury (increase of serum creatinine >0.5 mg/dl) and NGAL levels was determined using logistic and linear regression models. Results: 51 of 205 patients (25%) who received aprotinin developed acute kidney injury compared to 19 of 164 patients (12%) who received epsilon amino-caproic acid (p = 0.0013). Aprotinin use was associated with a two-fold higher risk of acute kidney injury when adjusted for potential confounders (age, Parsonnet score, preoperative serum creatinine, cardiopulmonary bypass and cross-clamp times; multiple logistic regression: OR = 2.164; CI (95%) = 1.102 to 4.249; p = 0.0249. Urinary NGAL was 19 times higher immediately after cardiopulmonary bypass and 18 times higher 3 h later in patients who had received aprotinin (postoperative: 19.23; CI (95%) = 12.60 to 29.33; p < 0.0001; 3 h post-cardiopulmonary bypass 18.67; CI (95%) = 11.45 to 30.43; p < 0.0001). Conclusions: Postoperative urinary NGAL – a novel marker for renal injury – is increased in cardiac surgical patients receiving aprotinin compared to patients receiving epsilon amino-caproic acid. These results further support the hypothesis that aprotinin may cause renal injury. The substantial rise of urinary NGAL associated with aprotinin use may in part be due to aprotinin blocking the uptake of NGAL by megalin/gp330 receptors in the proximal tubules.