17α-Boldenone (17α-BOL) and/or 17β-boldenone (17β-BOL) appear occasionally in fecal matter of cattle. In addition to 17α-BOL, a whole array of boldenone related substances can be found in the same samples. In vitro experiments with microsomal liver preparations and isolated hepatocytes combined with the excretion profiles found in urine and feces samples of in vivo experiments made it possible to identify several metabolites of 17β-BOL in 17β-BOL positive feces samples. In one animal treated with 17β-BOL, no 17β-BOL or its metabolites were present before treatment and most of these compounds disappeared gradually in time after the treatment was stopped. It is not clear what the origin is of 17α-BOL and boldenone metabolites in samples screened routinely for the abuse of anabolic steroids and considered to be ‘negative’ because of the absence of 17β-BOL since other workers showed some evidence that 17α-BOL can be of endogenous origin. However, in our hands, most of these 17α-BOL positive samples, obtained during routinely performed screenings of cattle, contained large amounts of Δ⁴-androstene-3,17-dione (AED), which normally is absent from routinely screened negative samples. Furthermore, AED was absent in all samples obtained from the animals treated with 17β-BOL. We have no direct evidence that 17α-BOL or 17β-BOL is of endogenous origin.