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Per- and poly-fluoroalkyl substances (PFAS) are ubiquitously distributed throughout aquatic environments and can bioaccumulate in organisms. We examined dietary uptake and depuration of a mixture of 3 PFAS: perfluorooctanoic acid (PFOA; C 8 HF 15 O 2 ), perfluorooctane sulfonate (PFOS; C 8 HF 17 SO 3 ), and hexafluoropropylene oxide dimer acid (HPFO-DA; C 6 HF 11 O 3 ; trade name GenX). Benthic fish (blue spot gobies, Pseudogobius sp.) were fed contaminated food (nominal dose 500 ng g -1 ) daily for a 21-d uptake period, followed by a 42-d depuration period. The compounds PFOA, linear-PFOS (linear PFOS), and total PFOS (sum of linear and branched PFOS) were detected in freeze-dried fish, whereas GenX was not, indicating either a lack of uptake or rapid elimination (<24 h). Depuration rates (d -1 ) were 0.150 (PFOA), 0.045 (linear-PFOS), and 0.042 (linear+branched-PFOS) with corresponding biological half-lives of 5.9, 15, and 16 d, respectively. The PFOS isomers were eliminated differently, resulting in enrichment of linear-PFOS (70-90%) throughout the depuration period. The present study is the first reported study of GenX dietary bioaccumulation potential in fish, and the first dietary study to investigate uptake and depuration of multiple PFASs simultaneously, allowing us to determine that whereas PFOA and PFOS accumulated as expected, GenX, administered in the same way, did not appear to bioaccumulate. Environ Toxicol Chem 2020;39:595-603. © 2019 SETAC., (© 2019 SETAC.)