11-Oxoetiocholanolone and related substances are important metabolites of cortisol and are excreted via feces in ruminants. To investigate whether 11-ketotestosterone (11-KT) or its immunoreactive metabolites are formed and excreted in ruminant feces, an enzyme immunoassay (EIA) was developed and validated. The antibody was raised in rabbits against 11-KT-3-CMO:bovine serum albumin with biotinylated 11-KT as a label. The assay showed a sensitivity of 0.3pg/well. To validate the assay biologically, 6 rams were injected with a synthetic analogue of the adrenocorticotropic hormone (Synacthen, 2μg/kg body wt). An aliquot was collected of each fecal portion spontaneously defecated 8h before Synacthen injection to 24h after injection and stored at -20°C until analysis. Samples (0.5g) were extracted using 80% methanol and immunoreactive metabolites measured using the 11-KT EIA and an already established 11,17-dioxoandrostane (11,17-DOA) EIA. High-performance liquid chromatography separation revealed no peak in the same elution position as authentic 11-KT; therefore, reacting substances were referred to as 11-KT equivalents. In the case of 11-KT immunoreactive substances, the values increased from baseline (median, 136ng/g feces) to a peak concentration (median, 424ng/g) 10 to 14h after Synacthen injection and declined afterwards. Concentrations of 11,17-DOA showed the same pattern, but the values were 2 to 4 times higher. From this data, the authors conclude that 11-KT-like substances, specifically C19O3-androgens with a 17ß-hydroxy group, were present in the feces. These substances originate from the adrenals and are most likely cortisol metabolites. © 2013 SETAC.