In several studies, hormones such as gonadotropin-releasing hormone (GnRH), prostaglandins and oxytocin were used to increase pregnancy rate by inducing ovulation and improving the sperm transport in the female reproductive tract in lactating dairy cattle. The objective of this study was to compare the effects of GnRH, prostaglandin F-2 alpha (PGF(2 alpha)) and oxytocin treatments at the time of artificial insemination (AI) after spontaneous oestrus on the conception rate (CR) of lactating dairy cows. Oestrus was detected by visual observations by experienced personnel. All cows (n = 430, 308 Holstein-Frisian and 122 Swedish-Red dairy cows) were inseminated based on the am/pm rule by veterinarians of the farm. After AI, cows were alternately assigned to one of the four treatment groups: (1) GnRH (n = 113); (2) PGF(2 alpha) (n = 106); (3) oxytocin (n = 106) and (4) non-treated control (n = 105). Pregnancy diagnosis was performed 28-34 and 58-64 days post-insemination by transrectal ultrasonography. Conception rates on days 28-34 and 58-64 were not different among GnRH (46.0%; 52/113 and 44.3%; 50/113), PGF(2 alpha) (37.7%; 40/106 and 35.9%; 38/106) and control (49.5%; 52/105 and 47.6%; 50/105) groups. However, conception rates were lower (P = 0.02) in oxytocin (31.1%; 33/106 and 30.2%; 32/106) than in GnRH and control groups on days 28-34 and 58-64. Other covariant factors, such as milk production, days in milk (DIM), breed, parity, service number did not affect the conception rate. Thus, there were no beneficial effects of treatments with GnRH and PGF(2 alpha) at the time of AI, and oxytocin had an adverse effect on CR in lactating dairy cows in this study.