A method for the detection of ethinylestradiol in cattle urine is described, based on enzymic hydrolysis of the sample, clean-up by means of disposable octadecyl and amino solid-phase extraction columns, fractionation by reversed-phase high-performance liquid chromatography, and detection by gas chromatography-mass spectrometry (selected-ion monitoring). Identification is based on both gas chromatographic and mass spectrometric data. The method has been tested on urine samples for a collaborative study and all the results found were correct.