A gas chromatography-tandem mass spectrometry (GC-MS(2)) method for the detection and quantification of 17alpha-ethinylestradiol in the hair of cattle has been developed, and uses an ion trap analyzer. After the digestion of 500 mg of hair by alkaline digestion using 1 M NaOH, extraction and purification of the steroid were performed in the same step by means of diphasic dialysis. This technique is a semipermeable-membrane technology developed for the direct extraction of relatively low-molecular-mass analytes. The process was performed by employing acetate buffer to homogenize the digested hair, dichloromethane as the extraction solvent at 37 degrees C, and stirring at 150 rpm for 4 h. The recovery was between 74 and 94%. The detection limit was 0.52 ng/g in hair. To evaluate the validity of the methodology, five animals, approximately 3 months old, received an intramuscular anabolic dose of the drug. The xenobiotic could be detected 7 or 14 days after the treatment (between 2.01 and 23.61 ng/g), and until the end of the study (day 98). No statistical difference between hair color and hair assay outcomes was found.