Nortestosterone (β‐NT) is a hormonal growth promoter banned from livestock production in the EU. Following injection, the major metabolite in cattle is the 17α‐epimer (α‐NT). However, this also occurs naturally in pregnant cattle. It is not known whether α‐NT is also endogenous to intact or castrated male cattle. Three surveys were undertaken to assess whether α‐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 α‐NT were subjected to GC‐MS. With 2 ng/ml α‐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 β‐NT, did not have measurable concentrations of α‐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 α‐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 α‐NT higher than the limit of quantification of the assay LOQ (0.7 ng/ml). Of these animals, two had β‐NT‐containing injection sites and five had residues of the β‐agonists clenbuterol and mabuterol. Examination of the animal movement and ownership histories of the 56 confirmed positivetanimals strongly suggested that exogenous 13‐NT had been administered at the presenting farm. It is concluded that α‐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.