Biliary excretion rate of [14C]taurocholate was 53–63% greater in rainbow trout (Salmo gairdneri) acclimated to and tested at 18 than 14 or 10 °C. Acute 4° temperature shifts up or down increased or decreased respectively biliary excretion rate by 40–53%. Furthermore, 10 °C acclimated fish shifted to 14 °C had 125% greater biliary excretion rate than 18 °C acclimated fish shifted to 14 °C. Apparent hepatic blood flow was greater in fish acclimated to and measured at 18 than 14 or 10 °C but was not different when 10 or 18 °C acclimated fish were measured at 14 °C. Acclimation to 10 versus 18 °C did not affect the Vmax or the temperature/activity relationship for the liver plasma membrane (LPM) Na+,K+-ATPase or the LPM Mg2+ -ATPase but did cause a major shift in the Km of the Na+,K+-ATPase (0.14 and 1.11 mM, respectively). This Km shift was observed at an 18 °C but not a 37 °C assay temperature. Thus, acclimation-induced changes in this enzyme were only observable at physiologically relevant temperature and substrate conditions. Depending on the thermal treatment, both hepatic blood flow and LMP Na+,K+-ATPase may module biliary excretion.