Background: Iatrogenic ureteric injury remains a risk in laparoscopic pelvic procedures. Near-infrared fluorescence (NIRF) imaging is a promising new technique for enhanced intraoperative visualization of anatomical structures that could improve the safety of laparoscopic surgery. A new dye, IRDye (R) 800-BK, has been developed for intraoperative visualization of the ureters using NIRF. The present study was a first evaluation of the performance of IRDye (R) 800-BK for ureteric imaging during NIRF laparoscopy. Methods: This study consisted of three parts: real-time in vivo NIRF imaging using IRDye (R) 800-BK in pigs during laparoscopic surgery, ex vivo NIRF imaging of freshly explained pig ureters and ex vivo NIRF imaging of explanted human ureters. Results: In all animals, both left and right ureters were visualized throughout the laparoscopic procedure for 120 min, with the best results at a dose of 0.15 mg dye per kg bodywcight. NIRF imaging was successful in all human and porcine ureters studied, with a range of dye concentrations. Conclusion: NIRF imaging of the ureters using IRDye (R) 800-BK was used successfully both in vivo in a porcine model, and ex vivo in porcine and human ureters.