The development of novel methods to facilitate the indirect detection of the illegal administration of sex steroid hormones and other growth promoters would enhance the efficiency and success rate of food screening and safety programmes established by state authorities. Particularly, the transcriptomic approach could facilitate the identification of biomarkers suitable for the detection of illegally treated animals. Recent studies have shown that progesterone receptor gene expression levels were increased in the bulbo-urethral and prostate glands of 17b-estradiol-treated calves and beef cattle. For potential use in food safety monitoring, a quantitative PCR method has been developed for the detection of upregulated PR gene expression in the bulbo-urethral glands of beef cattle and veal calves illegally administered 17b-estradiol. Currently, there are no studies focusing on RGN expression in bovine tissues and organs. The first aim of the present study was to investigate RGN gene and protein expression in different tissues of veal calves and beef cattle. We also determined whether RGN expression is controlled through sex steroid hormones in bovine target tissues, namely the bulbo-urethral and prostate glands and the testis. The significant change in RGN gene expression may well be an intriguing biomarker to discover hormone abuse in bovine husbandry. The described methodology, using an indirect marker to detect illegal hormone treatment, promises to significantly improve food safety control programs once introduced. Although several studies have investigated RGN function in different species, the RGN expression in bovine tissues and organs has not been explored. To our knowledge, this study is the first to report RGN mRNA expression in bovine organs and tissues other than the liver. WB analysis using an anti-RGN polyclonal antibody showed a reactive immunoprotein of approximately 33 kDa, corresponding to the predictive size of RGN. The IHC analyses also confirmed the RGN expression in the accessory sex glands, where the protein is localised to both the cytoplasm and nuclei of cells. Indeed, RGN translocates from the cytoplasm to the nucleus where this protein regulates DNA synthesis and fragmentation, the expression of oncogenes, tumour suppressor genes and cell cycle regulators. Particularly, this cellular localisation pattern suggests a relevant role in testicular physiology in both veal calves and beef cattle. RGN is an important regulator of cellular Ca2+ homeostasis in several tissues. Ca2+ serves important biological functions, acting as a second messenger in several transduction pathways or regulating apoptotic cell death, among others. A tightly regulated equilibrium between germ cell apoptosis and proliferation is required for a successful spermatogenesis, as approximately 75% of testicular germ cells undergo apoptosis. Moreover, the tight control of intracellular Ca2+ homeostasis is critically important in the maintenance of Sertoli cell function and Leydig cell steroidogenesis. The histological evaluation of the testis from veal calves treated with BAY 73-4506 17b-estradiol or testosterone showed an interruption of germ cell line development, as previously described.