To date, research with isolated perfused fish livers has been limited by the relatively short time period during which stable performance can be achieved. In the present study, modifications to existing methods were employed with the goal of extending the usable life of an isolated perfused trout liver preparation. Liver performance was evaluated by measuring O 2 consumption (VO 2 ), vascular resistance, K + leakage, glucose flux, lactate flux, and clearance of a model metabolic substrate, 7-ethoxycoumarin (CL H,7-EC ). Livers perfused with solutions containing 15, 38, or 150μM bovine serum albumin (BSA) exhibited relatively stable physiological performance for up to 10h. CL H,7-EC decreased rapidly between 1 and 2h in all livers tested, possibly due in part to accumulation of 7-EC within the tissue. CL H,7-EC declined slowly thereafter, decreasing by 30–40% between 2 and 10h. A linear equation was subsequently developed to correct measured levels of clearance for this decrease in metabolic activity over time. To illustrate the value of this preparation, experiments were conducted to examine the effects of protein binding on 7-EC clearance. Clearance rates corrected for declining activity (CL H,7-EC,CORR ) changed in nearly direct proportion to changes in the free concentration of 7-EC efferent to the liver, as predicted by theoretical models of liver function. Additional studies were performed to characterize the concentration-dependence of 7-EC clearance. The rate of substrate disappearance from the perfusate increased in proportion to the total concentration of 7-EC afferent to the liver resulting in constant levels of CL H,7-EC,CORR . CL H,7-EC,CORR values for four livers averaged 12.1±2.5mL/h/g-liver (mean±SD, n=57 individual determinations) and were in good agreement with an estimate of hepatic clearance obtained by extrapolating published in vitro data from isolated trout hepatocytes. The extended viability of isolated trout livers achieved in this study creates new opportunities for research on hepatic function in fish.