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Background: Drinking water contamination related to the use of aqueous film-forming foam (AFFF) has been documented at hundreds of military bases, airports, and firefighter training facilities. AFFF has historically contained high levels of long-chain per- and polyfluoroalkyl substances (PFAS), which pose serious health concerns. However, the composition and toxicity of legacy AFFF mixtures are unknown, presenting great uncertainties in risk assessment and affected communities., Objectives: This study aimed to determine the fluorinated and nonfluorinated chemical composition of a legacy AFFF sample and its toxicity in zebrafish embryos., Methods: A sample of legacy AFFF (3% application formulation, manufactured before 2001) was provided by the Massachusetts Department of Environmental Protection. High resolution mass spectrometry (HRMS) was used to identify PFAS and nonfluorinated compounds, and a commercial laboratory measured 24 PFAS by a modified U.S. EPA Method 537.1. AFFF toxicity was assessed in zebrafish embryos in comparison with four major constituents: perfluorooctanesulfonic acid (PFOS); perfluorohexanesulfonic acid (PFHxS); sodium dodecyl sulfate (SDS); and sodium tetradecyl sulfate (TDS). End points included LC 50 values, and sublethal effects on growth, yolk utilization, and pancreas and liver development., Results: We identified more than 100 PFAS. Of the PFAS detected, PFOS was measured at the highest concentration ( 9,410 mg / L ) followed by PFHxS ( 1,500 mg / L ). Fourteen nonfluorinated compounds were identified with dodecyl sulfate and tetradecyl sulfate the most abundant at 547.8 and 496.4 mg / L , respectively. An LC 50 of 7.41 × 10 - 4 % AFFF was calculated, representing a dilution of the 3% formulation. TDS was the most toxic of the constituents tested but could not predict the AFFF phenotype in larval zebrafish. PFOS exposure recapitulated the reduction in length but could not predict effects on development of the liver, which was the tissue most sensitive to AFFF., Discussion: To our knowledge, this research is the first characterization of the chemical composition and toxicity of legacy AFFF, which has important implications for regulatory toxicology. https://doi.org/10.1289/EHP6470.