A high level of14C was found to bind irreversibly with the liver proteins of rainbow trout (Oncorhyncus mykiss) exposed to 135 mg/kg bodyweight of14C‐labelled furazolidone (14C‐FZ) in fish feed daily for 10 days. After the cessation of14C‐FZ treatment, hepatic protein‐bound14C in trout stayed high for at least 30 days. The chemical identity of protein‐bound14C remained to be elucidated. However, a part of the protein‐bound14C in the liver and muscle could be released as 3‐amino‐2‐oxazolidinone by acid hydrolysis. The formation of protein‐boundI4C in the liver was investigated further with trout dosed intravenously with different14C‐FZ doses at 10°C or with 5 mg/kg FZ at different water temperatures.14C binding to the liver proteins was found to increase with increasing FZ dose or water temperature. Results of these studies indicate that protein‐bound14C in the muscle and liver of trout is related to the formation of reactive intermediates from FZ. However, additional studies on the identities of the protein‐bound FZ residues are required before they can serve as useful biomarkers to monitor FZ exposure in farm fish.