The aim of this study was to implement the progesterone EIA test, developed in our laboratory by using an anti-progesterone antibody (Yamaguchi University, Japan), in order to determine the optimal moment for artificial insemination (AI) and to detect pregnancy in Holstein-Friesian cows according to the progesterone concentration in the whole milk. Also, the influence of β-carotene, applied at the day of insemination and human chorionic gonadotrophin applied on day 7 after AI on the progesterone level and the pregnancy rate were evaluated.For the accuracy of oestrus detection, the milk samples from 70 cows were collected on the day of insemination. Milk samples from 148 cows were collected 19-22 days following insemination for pregnancy check. After detection of naturally occurring oestrus (day 0) and AI, cows were divided into the following groups: group A (n = 19) was treated with 200 mg β-carotene (20 ml Carofertin® i.m. Alvetra u. Werfft Gmbh, Austria), group B (n = 17) was treated with 1 500 IU hCG i.m. (Schering-Plough, the Netherlands) and  control (non treated) group C (n = 18). The milk samples for EIA progesterone concentration analysis were collected on the day of AI, the 14th and the 20th day of the oestrus cycle. Oestrus detection errors and inappropriate moments of insemination according to the progesterone concentration were detected in 22.86% animals (16/70). The test accuracy for non-pregnant cows was 90.48% (76/84). The accuracy of the progesterone test in pregnant cows was 75% (48/64). False positive results (high progesterone level, but the cows were not pregnant) was detected in 25% of cows (16/64) as a result of a prolonged oestrus cycles, embrional mortality and endomethritis (10/16 cases). The treatment of cows with 1500 IU of hCG, on the day 7 of the oestrus cycle, resulted in statistically significant increase of progesterone concentration in the dioestrus (P < 0.01). The most successful insemination was in the group of cows that was treated with hCG (47.05%; 8/17), then in the control group (38.88%; 7/18) and the least in the Carofertin group - 36.84% (7/19). These differences were only numerical (p>0.05). The EIA test developed in our laboratory could be used for accurate progesterone determination in the whole milk during implementation of different methods for control of bovine reproduction.