In a preliminary trial and three experiments, a total of 30 Holstein heifers were experimentally infected with a culture of Leptospira borgpetersenii serovar hardjobovis via one or more routes (uterine, cervical supraconjunctival, intranasal) and oviductal and uterine fluids recovered post-mortem or in vivo following superovulation with FSH. All routes of administration were effective in establishing Leptospira infection in the reproductive tract and Leptospira were identified in the oviductal and uterine fluids of all 30 heifers by microscopy. The incidence of infection was confirmed by positive identification of serum antibodies by the microscopic agglutination test (MAT). Twenty-one samples of the embryos (n = 59) recovered were cultured using bacteriological procedures and all tested negative for the infectious microorganism. Using polymerase chain reaction (PCR) assay, however, showed that 29% (7/24) of morula and blastocyst stage embryos, and one out of 29 oocytes tested positive for the presence of leptospiral DNA. A single oocyte or embryo collected from the infected heifers was inoculated intravenously to 26 test heifers. None of the test heifers developed antibody titers to Leptospira. It was concluded that, despite the presence of leptospires in the reproductive tract of donor animals and the association of leptospiral DNA with uterine stage embryos, the transmission of this disease is unlikely to occur by transfer of in vivo produced embryos in the bovine.