The effects of fasting and feeding on the activity and kinetics of the hepatic microsomal L-thyroxine (T4) 5′-monodeiodinase enzyme system (5′D) responsible for generating 3,5,3′-triiodo-L-thyronine (T3) were studied in immature rainbow trout held on a 12 h L: 12 h D photocycle at 12 °C. The 5′D activity was determined from the essentially stoichiometric production of 125I− and 3,5,[3′-125I]T3 as the primary labeled materials from 3,5,[3′-125I],5′-T4 substrate, as judged by LH-20 Sephadex chromatography and HPLC. Relative to trout fed 2% body weight∙day−1 (2% ration), fasting depressed 5′D activity within 3 days. By 7 days a basal 5′D level was reached, which persisted for at least 20 days. In trout fed a 0.8% ration, fasting for 8 days depressed 5′D activity to a level lower than that of trout fed a 2% prefasting ration, suggesting 5′D response to energy reserves. However, following 8 days fasting, refeeding for 7 days reinstalled 5′D activity, regardless of prior maintenance on a 0.8 or 2% ration. There was a strong positive correlation between body weight gain and 5′D activity for trout fed rations of 0, 0.3, 0.6, 1, 2, or 3% for 14 days. Kinetic analyses showed that change in 5′D activity was due mainly to change in Vmax (quantity of functional enzyme units). However, fasting also decreased Km (Michaelis–Menten constant), suggesting the presence of a 5′D form with a higher T4 affinity. The 5′D activity was significantly correlated with the blood plasma T3 level, but not with the plasma T4 level. We conclude that (i) hepatic 5′D responds rapidly to both food deprivation and feeding and is also sensitive to energy reserves, (ii) the response primarily involves the level of functional 5′D units but some change in enzyme form may also occur, and (iii) the high positive correlations between 5′D activity, plasma T3 level, and gain in body weight are consistent with a role of hepatic 5′D in generating systemic T3, which in turn may regulate somatic growth.