An analytical method has been developed and validated for the simultaneous trace determination of four macrolide antibiotics, six sulfonamides, the human metabolite N4-acetylsulfamethoxazole, and trimethoprim in wastewater. The method was validated for tertiary, secondary, and-unlike in previously published methods-also for primary effluents of municipal wastewater treatment plants. This wide range of application is necessary to thoroughly investigate the occurrence and fate of chemicals in wastewater treatment. Wastewater samples were enriched by solid-phase extraction, followed by reversed-phase liquid chromatography coupled to tandem mass spectrometry using positive electrospray ionization. Recoveries from all sample matrixes were generally above 80%, and the combined measurement uncertainty varied between 2 and 18%. Concentrations measured in tertiary effluents ranged between 10 ng/L for roxithromycin and 423 ng/L for sulfamethoxazole. Corresponding levels in primary effluents varied from 22 to 1450 ng/L, respectively. Trace amounts of these emerging contaminants reach ambient waters, since all analytes were not fully eliminated during conventional activated sludge treatment followed by sand filtration. In the case of sulfamethoxazole, the amount present as human metabolite N4-acetylsulfamethoxazole had to be taken into account in order to correctly assess the fate of sulfamethoxazole in wastewater treatment.