Leptospirosis, a disease acquired by exposure to contaminated water, is characterized by fever accompanied by various symptoms, including abdominal pain. An acute febrile illness occurred in athletes who participated in an Illinois triathlon in which the swimming event took place in a freshwater lake. Of 876 athletes, 120 sought medical care and 22 were hospitalized. Two of the athletes had their gallbladders removed because of abdominal pain and clinical suspicion of acute cholecystitis. We applied an immunohistochemical test for leptospirosis to these gallbladders and demonstrated bacterial antigens staining (granular and filamentous patterns) around blood vessels of the serosa and muscle layer. Rare intact bacteria were seen in 1 case. These results show that leptospirosis can mimic the clinical symptoms of acute cholecystitis. If a cholecystectomy is performed in febrile patients with suspicious environmental or animal exposure, pathologic studies for leptospirosis on formalin-fixed, paraffin-embedded tissues may be of great value.