OBJECTIVE: Anatomic identification of parathyroid glands during surgery is challenging and time consuming. We sought to determine whether 5-aminolevulinic acid (5-ALA) could produce parathyroid gland fluorescence to improve their detection in a preclinical model. METHODS: Thirty-two rats were administered 0 to 700 mg/kg of 5-ALA by intraperitoneal injection prior to neck exploration under the illumination of a blue light (380-440 nm). Tissue fluorescence was assessed at 1, 2, or 4 hours postinjection and then removed for histologic confirmation of parathyroid tissue. RESULTS: Rat parathyroid glands could not be visualized under ambient light. At dosages of 300 mg/kg or greater, bilateral parathyroid glands were visualized in 18 of 19 rats using blue light illumination. At dosages less than 300 mg/kg, parathyroid gland fluorescence was detected in only 1 of 13 rats. At 2 hours after 5-ALA administration, the net mean intensity of parathyroid gland fluorescence was optimal with a dose of 500 mg/kg. At both 1 and 4 hours after 5-ALA injection, the net mean intensity of parathyroid gland fluorescence was optimal at the highest dose (700 mg/kg) and positively correlated with dosage increases. CONCLUSION: 5-ALA can be used to selectively detect parathyroid tissue from surrounding tissue in a preclinical model. Our data support the use of this technique in the clinical setting. © The American Laryngological, Rhinological & Otological Society, Inc.