Metabolism plays an important role in bioaccumulation of xenobiotics in fish. The applicability of trout liver microsomes and S9 fraction in bioaccumulation assessment of xenobiotics in fish was investigated in the present study. Basal-level activities of 7-ethoxyresorufin-O-dealkylase, testosterone 6β-hydroxylase, glutathione-S-transferase, and uridine 5′-diphospho-glucuronosyl-transferase in trout liver microsomes and S9 were significantly lower than those in rat liver microsomes and S9. The in vitro–to–in vivo scaling factors, which are the values of liver microsomal and S9 protein contents per unit weight of trout liver, were determined to be 38.4 ±5.1 (mean ± standard deviation throughout) and 95.9 ±11.9 mg/g, respectively. Intrinsic clearance (CLint) values for a number of reference compounds obtained from trout liver S9 were lower than those from trout liver microsomes. After correction with the scaling factors, trout liver microsomes and S9 provided equivalent prediction of trout hepatic clearance (CLH) using the well-stirred liver model, but their CLH values were significantly lower than those obtained from freshly isolated trout hepatocytes. Consequently, trout liver microsomes and S9 showed poorer prediction of the bioconcentration factors of the reference compounds compared with trout hepatocytes. Unit conversion revealed that CLint values obtained from trout liver microsomes and S9 were 6.3 to 22.4% of those from trout hepatocytes, which explained, to a large extent, the differences in their CLH and bioconcentration factor prediction.