A lifetime controlled reference experiment has been performed using 42 veal calves, 21 males and 21 females which were fed and housed according to European regulations and common veterinary practice. During the experiment feed, water, urine and hair were sampled and feed intake and growth were monitored. Thus for the first time residue analysis data were obtained from guaranteed lifetime-untreated animals. The analysis was focused on the natural hormones estradiol and testosterone and their metabolites, on 17beta- and 17alpha-nortestosterone, on 17beta- and 17alpha-boldenone and androsta-1,4-diene-3,17-dione (ADD), and carried out by gas chromatography tandem mass spectrometry (GC/MS/MS), an estrogen bioassay and liquid chromatography (LC) MS/MS. Feed, water and hair samples were negative for the residues tested. Female calf urines showed occasionally low levels of 17alpha-estradiol and 17alpha-testosterone. On one particular sampling day male veal calf urines showed very high levels of 17alpha-testosterone (up to 1000 ng mL(-1)), accompanied by lower levels of estrone and 17beta-testosterone. Despite these extreme levels of natural testosterone, 17beta-boldenone was never detected in the same urine samples; even 17alpha-boldenone and ADD were only occasionally beyond CCalpha (maximum levels 2.7 ng mL(-1)). The data from this unique experiment provide a set of reference values for steroid hormones in calf urine and demonstrate that 17beta-boldenone is not a naturally occurring compound in urine samples.