Bioconcentration factors (BCFs) in rainbow trout were measured for 10 anionic surfactants with a range of alkyl chain lengths and different polar head groups. The BCFs ranged from 0.04 L kg^–1 ww (for C₁₀SO₃) to 1370 L kg^–1 ww (C₁₆SO₃). There was a strong correlation between the log BCF and log membrane lipid–water distribution ratio (D_MLW, r² = 0.96), and biotransformation was identified as the dominant elimination mechanism. The strong positive influence of D_MLW on BCF was attributed to two phenomena: (i) increased partitioning from water into the epithelial membrane of the gill, leading to more rapid diffusion across this barrier and more rapid uptake, and (ii) increased sequestration of the surfactant body burden into membranes and other body tissues, resulting in lower freely dissolved concentrations available for biotransformation. Estimated whole-body in vivo biotransformation rate constants k_B-BCF are within a factor three of rate constants estimated from S9 in vitro assays for six of the eight test chemicals for which k_B-BCF could be determined. A model-based assessment indicated that the hepatic clearance rate of freely dissolved chemicals was similar for the studied surfactants. The dataset will be useful for evaluation of in silico and in vitro methods to assess bioaccumulation.