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Ohio’s oral rabies vaccination (ORV) program was established to prevent the westward spread of the raccoon (Procyon lotor) rabies virus (Lyssavirus, Rhabdoviridae) in Ohio, USA. The program, which targets raccoons, distributes vaccine-bait units (VBU) at a target density of 75 units/km2. Few studies have examined the relationship of VBU density and target population density to the prevalence of rabies virus-neutralizing antibodies (RVNA). We conducted experimental VBU distributions in August 2003 and August 2004, 150 km west of the ORV zone where there was no history of raccoon rabies. We measured change in RVNA titers in blood collected from live-trapped raccoons before and after VBU distributions. A closed population mark–recapture estimate of the size of the target population was 91 raccoons/km2, compared to the realized VBU distribution density of 70 units/km2. Surprisingly, 41% of 37 serum samples were RVNA-positive (≥0.05 IU/ml) before VBU distribution in 2003, but all titers were 2 was insufficient to produce a population-wide immunoprotective response against rabies infection in our high-density target population. Presence of RVNA in a presumed naive population before baiting demonstrates that estimating prevalence of RVNA after oral rabies vaccination can be problematic without knowledge of background titers and seasonal changes in prevalence of RVNA before and after baiting.