Abstract The bioaccumulation behavior, including the uptake, internal distribution, depuration, and biotransformation rates, of three widely used linear methyl-siloxanes was investigated in rainbow trout. Dietary uptake efficiencies of octamethyltrisiloxane (L3), decamethyltetrasiloxane (L4), and dodecamethylpentasiloxane (L5) were 15% (3.3% standard error [SE]), 8.6% (1.4% SE), and 15% (1.8% SE), respectively, and for L3 and L4 were well below those of nonmetabolizable reference chemicals with similar octanol–water partition coefficients, suggesting significant intestinal biotransformation of L3 and L4. Somatic biotransformation rate constants were 0.024 (0.003 SE) day–1 for L3 and 0.0045 (0.0053 SE) day–1 for L4 and could not be determined for L5. Lipid-normalized biomagnification factors for L3, L4, and L5 were 0.24 (0.02 SE), 0.24 (0.01 SE), and 0.62 (0.05 SE) kg-lipid kg-lipid–1, respectively. Bioconcentration factors standardized to a 5% lipid content fish for water in Canadian oligotrophic lakes with a dissolved organic carbon content of 7.1 mg L–1 were 2787 (354 SE) for L3, 2689 (312 SE) for L4, and 1705 (418 SE) L kg-wet weight–1, respectively, and 3085 (392 SE) for L3, 4227 (490 SE) for L4, and 3831 (938 SE) L kg-wet weight–1 in water with a dissolved organic carbon content of 2.0 mg L–1. A comparison of 238 bioaccumulation profiles for 166 different chemicals shows that the bioaccumulation profiles for L3, L4, and L5 are vastly different from those of other very hydrophobic compounds found in the environment. Environ Toxicol Chem 2024;43:42–51. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.