The object of this study was to isolate and chemically characterize the most acutely toxic fraction of a petroleum refinery wastewater. Daphnia magna static bioassays were used to characterize the relative toxicity of each fraction. Steam stripping the wastewater concentrated the toxicity in a volatile fraction. Activated carbon filtration removed the toxicity but cation exchange did not, indicating that organics are the toxic agents. Methylene chloride extraction of the volatile portion at pH > 11 followed by silica gel column chromatography resulted in an aromatic residue which was acutely toxic when dissolved in culture water. The combination of selected fractionation followed by Daphnia bioassays of each fraction determined the characteristics of the most toxic fraction to be steam volatile, base-neutral, and aromatic. Eleven compounds (polycyclic aromatic hydrocarbons) with a total concentration of 1100 µg/L and accounting for 28% of the total peak area of the chromatogram were identified in the aromatic fraction by a gas chromatograph/mass spectrometer (GC/MS). The mass spectra of the unidentified compounds indicated that they were probably nonhalogenated heterocyclics with molecular weights of 180 to 300, containing nitrogen, oxygen, or sulfur atoms. Some of the unidentified compounds appeared to be hydroxylated forms of the parent compounds which were identified.