The aims of this study were to assess whether thermal tolerance of brook trout (Salvelinus fontinalis) is affected during seawater (SW) acclimation and to investigate the role of cortisol in osmoregulation and thermal tolerance during SW acclimation. Freshwater (FW)-acclimated brook trout at 18°C (T_acc) were exposed to SW for 16 days, whilst maintaining a FW control. Fish were examined for critical thermal maximum (CT_max) 0 (before), 2, 5 and 16 days after SW exposure, and sampled at T_acc and CT_max for analysis of plasma cortisol, glucose and Cl⁻, gill Na⁺/K⁺-ATPase (NKA) activity and heat shock protein 70 (HSP70) abundance, and white muscle water content. At 2 days in SW, CT_max was significantly reduced (from 31 to 26°C), and then recovered by 16 days. This transient decrease in thermal tolerance coincided with a transient increase in plasma Cl⁻ and decrease in muscle moisture content. Salinity itself had no effect on gill HSP70 abundance compared with the large and immediate effects of high temperature exposure during CT_max testing. To examine the role of cortisol in osmoregulation, brook trout were administered a cortisol implant (5 and 25 μg g⁻¹ CORT) prior to SW exposure. Both CORT doses significantly increased their capacity to maintain plasma Cl⁻ during SW acclimation. Treatment with the 25 μg g⁻¹ CORT dose was shown to significantly improve CT_max after 2 days in SW, and CT_maxwas associated with plasma Cl⁻ and muscle moisture content. These findings indicate that brook trout are sensitive to temperature during SW acclimation and that thermal tolerance is associated with ion and water balance during SW acclimation.