Nortestosterone (beta-NT) is a hormonal growth promoter banned from livestock production in the EU. Following injection, the major metabolite in cattle is the 17 alpha-epimer (alpha-NT). However, this also occurs naturally in pregnant cattle. It is not known whether alpha-NT is also endogenous to intact or castrated male cattle. Three surveys were undertaken to assess whether alpha-NT is naturally produced in this subset of the population. Bile samples from a total of 1,281 cattle (73 bulls and 1,208 steers) from 366 herds were collected at slaughter and initially screened by using a semi-automated EIA with multi-analyte immunoaffinity chromatography (IAC) clean-up. Bile samples from a further 38 male cattle (10 bulls and 28 steers) were analysed by high-resolution gas chromatography-mass spectrometry (GC-MS) with IAC pretreatment. Only samples containing more than 2 ng/ml alpha-NT were subjected to GC-MS. With 2 ng/ml alpha-NT as a threshold for confirmatory testing, the false positive rate of the screening EIA was 1.8%. Bulls (n = 16) and steers (n = 179) from government farms (n = 2) and which were not treated with exogenous beta-NT, did not have measurable concentrations of alpha-NT in their bile. Bulls (n = 35) and steers (n = 606) taken from herds (n = 204) which had no previous history of illegal growth promoter abuse also did not have alpha-NT in their bile. Of 32 bulls and 451 steers of unknown treatment history sampled from herds (n = 160), 56 steers from 19 herds contained GC-MS confirmed concentrations of alpha-NT higher than the limit of quantification of the assay LOQ (0.7 ng/ml). Of these animals, two had beta-NT-containing injection sites and five had residues of the beta-agonists clenbuterol and mabuterol. Examination of the animal movement and ownership histories of the 56 confirmed positive animals strongly suggested that exogenous beta-NT had been administered at the presenting farm. It is concluded that alpha-NT is not endogenous to this subset of the cattle population and that detection of this hormone in bile from bulls and steers constitutes evidence of abuse.