The presence of virulent Newcastle disease virus (NDV) since the 1993-94 epidemic in southern Africa holds major implications for the export of ostrich products from this region. A challenge experiment with this field strain was conducted in open-air feedlot facilities under strict biosecurity measures. The experiment was designed to follow vaccination and preslaughter quarantine regulations currently enforced in South African export ostrich facilities in order to determine the viremia period and immune response under these specific circumstances. One hundred forty-three slaughter ostriches were allocated into three test groups, according to the time period between pretrial vaccination and challenge (1-2 mo, 2-4 mo, 4-6 mo), and an unchallenged control group. All birds in the test groups were challenged by oral, tracheal, and ocular routes with a field isolate of NDV. They were slaughtered over the next 4 wk on nine separate occasions and bled on 12 occasions. Virus isolation was attempted from seven sets of pooled samples from each bird to determine the viremia period and the serum antibody concentrations were measured by hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) methods to establish an immune response curve. NDV could be back-isolated only up to day 9 postinfection and from only six ostriches with poor immune response titers and corresponding to a rise in antibody levels above an indirect ELISA optical density reading of 0.33. Virus could be recovered only from brain and respiratory tract tissue. The HI test was less sensitive than the ELISA. Immune response curves did not differ significantly between the groups and peaked on day 14 post-infection. From these data, ELISA titers would appear to be a good indicator of the probability that an ostrich will be clinically infected after velogenic NDV challenge. These results also suggest that the current vaccination schedule enforced by the South African Veterinary Authorities results in protective immunity in up to 95% of slaughter ostriches from export approved facilities. The standard 30-day preslaughter quarantine period introduced as part of Crimean-Congo hemorrhagic fever virus control measures also appears sufficient to encompass the determined NDV viremia period of 9-11 days in slaughter ostriches.