Commonly used indices of fish thyroidal status are based on thyroxine (T4) secretion by thyroid tissue under control of the central brain-pituitary-thyroid axis. However, much of the control of the fish thyroid system also occurs in peripheral tissues, such as liver, by regulating T4 prohormone conversion to biologically active 3,5,3'- triiodothyronine (T3) or to biologically inactive 3,3',5'-triiodothyronine and by regulating T3 conversion to inactive 3,3'-diiodothyronine. These extrathyroidal conversions depend on a family of independently-regulated selenocysteine-containing microsomal deiodinases. We describe deiodination assays and evaluate their potential as biomarkers for exposure to chemicals that directly or indirectly disrupt thyroid hormone homeostasis or thyroidal status. We conclude that deiodination be included in a minimum suite of assays to detect xenobiotic effects on the fish thyroid system.