OBJECTIVES: Ureter injury is a serious complication of laparoscopic surgery. Current strategies to identify the ureters, such as placement of a ureteral stent, carry additional risks for patients. We hypothesize that the systemically injected near-infrared (NIR) dye IRDye800CW-CA can be used to visualize ureters intraoperatively. METHODS: Adult female mixed-breed pigs weighing 24 to 41 kg (n = 2 per dose) were given a 30, 60, or 120 μg/kg systemic injection of IRDye800CW-CA. Using the Food and Drug Administration-cleared Pinpoint laparoscopic NIR system, images of the ureter and bladder were captured every 10 minutes for 60 minutes after injection. To determine the biodistribution of the dye, tissues were collected for ex vivo analysis with the Pearl Impulse system. ImageJ software was used to quantify fluorescence signal and signal-to-background ratio (SBR) for the intraoperative images. RESULTS: The ureter was identified in all pigs at each dose, with peak intensity reached by 30 minutes and remaining elevated throughout the duration of imaging (60 minutes). The 60 μg/kg dose was determined to be optimal for differentiating ureters according to absolute fluorescence (>60 counts/pixel) and SBR (3.1). Urine fluorescence was inversely related to plasma fluorescence (R(2) = -0.82). Ex vivo imaging of kidney, ureter, bladder, and abdominal wall tissues revealed low fluorescence. CONCLUSION: Systemic administration of IRDye800CW-CA shows promise in providing ureteral identification with high specificity during laparoscopic surgery. The low dose required, rapid time to visualization, and absence of invasive ureteral instrumentation inherent to this technique may reduce complications related to pelvic surgery.