Five 18- to 24-month-old bulls were inoculated with either a cell suspension containing bovine immunodeficiency virus (BIV-FL112; 3 bulls) or a BIV-free cell suspension (2 bulls). Blood and semen specimens were collected once a week for 14 weeks, and seroconversion was confirmed by indirect immunofluorescent antibody (IFA) testing. The presence of BIV in blood and semen was determined by virus isolation and/or polymerase chain reaction (PCR) assays. Antibodies to BIV were detected in the 3 experimentally infected bulls as early as day post inoculation (DPI) 17, and levels peaked at DPI 37–58. BIV was isolated from the peripheral blood mononuclear cells (MNCs) of the infected bulls at DPI 9 (2 bulls) and DPI 23 (1 bull), and could be isolated from one animal up to DPI 65. PCR analysis of MNC DNA, using BIV pol gene primers, detected virus in all three of the experimentally infected bulls from DPI 9 until the termination of the experiment at DPI 98. Efforts to isolate a significant number of non-spermatozoal cells (NSC) by gradient separation from the semen of the experimentally infected bulls were unsuccessful. Two methods for the extraction of total NSC DNA from up to 2 ml of non-extended semen were employed; however, no BIV pol fragment was amplified from these DNA preparations. Additionally, 30 bulls from artificial insemination (AI) centers were evaluated for BIV infection by PCR. No amplification products were obtained from MNC DNA from the AI submissions using primer sets for both the BIV pol and env genes.