Physiological levels of sex hormones 17β-oestradiol and testosterone vary according to the type of animal, age, sex, breed, manner of holding, nutrition, stress and environmental factors. Those hormones are necessary for growth and development of the animal, their sexual maturity and reproductive function. Because of their anabolic effect they were used in intensive livestock industry as growth promoters, applied in food or as an implant. But because of their toxic, mutagenic, teratogenic and cancerous effects on animal and human, they are forbidden for using as a purpose of improving the quality of the products of animal origin. Today it is only allowed to use this hormones in therapeutic purpose when it is necessary to treat some reproductive problem or problem in pregnancy. Considering the constant development of new synthetic substances that could have anabolic effects and possible misuse, it is necessary to provide regular controls during fattening and to know physiological values of the levels of these hormones. The aim of this work, taking all of the above, was to investigate the physiological levels of the natural hormones 17β-oestradiol and testosterone in the blood of calves with different age, sex and breed on the area of Republic of Croatia. In this purpose we have randomly selected in Croatia 32 calves of both sexes (5 males and 27 females), aging 2-5 months, different breeds and categories (13 calves of Simmental breed, 2 calves of meat breed and 17 calves of Holstein-Friesian breed). During general clinical examination we none of visible clinical changes were observed or conditions which could indicate which could alter physiological levels of investigating hormones. In this regard blood samples were taken and analyzed for these hormone levels in Croatian veterinary institute, Zagreb, Croatia. The average level of 17β-oestradiol is 28.83 ± 10.95 ng/L, while the testosterone level was under 50 ng/L in 24 calves and above 50 ng/L in 8 calves. Considering that the concentrations were consistent with the physiological concentrations of other authors, the suspected illegal use of the anabolic agent in those examined calves was excluded.