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Nonylphenol (NP) is a degradation product of nonylphenol polyethoxylate surfactants and has been reported to occur in water and sediments from urban areas. Technical NP is composed of several structural isomers, and conventional gas chromatography-mass spectrometry techniques have tentatively identified up to 22 components. Isolation and characterization of individual isomers in a technical NP mixture is important, because of the differences in estrogenic and bioaccumulation potential among the isomers. In this study, comprehensive two-dimensional gas chromatography (GC x GC) combined with mass spectrometry (MS) enabled tentative identification of 102 components of NP from a technical mixture. GC x GC-MS was also used to quantify two NP isomers in river water samples. This is the first study to use GC x GC-MS to characterize NP isomers in technical mixtures and for quantitative analysis of NP in river waters.