A laboratory study was conducted to investigate the role of the route of triphenyl phosphate (TPP) entry on its aquatic bioavailability and acute biological effects. Three TPP treatments were used for exposures of fish and invertebrates. These consisted of TPP dosed directly into water with and without clean sediment and TPP spiked onto sediment prior to aqueous exposures. Results of static acute toxicity tests (no sediment) were 0.78 mg/L (96-h LC50) for bluegill, 0.36 mg/L (48-h EC50) for midge, and 0.25 mg/L (96-h EC50) for scud. At 24 h, the sediment (1.1% organic carbon)/water partition coefficient (Kp) for TPP was 112. Use of this partition coefficient model to predict the sediment-mediated reduction of TPP concentration in water during toxicity tests resulted in a value that was only 10% less than the nominal value. However, the required nominal concentration of TPP to cause acute toxicity responses in test organisms was significantly higher than the predicted value by the model for both clay and soil-derived sediment. Direct spiking of TPP to soil minimized TPP bioavailability. Data from parallel experiments designed to track TPP residues in water through time suggest that sorption kinetics control residue bioavailability in the initial 24 h of exposure and may account for observed differences in LC50 and EC50 values from the sediment treatments.