Between 1984 and 1988, a study was conducted to evaluate the frequency of rabies virus neutralizing antibodies in raccoons (Procyon lotor) in two counties in Iowa. Nine hundred eighty five raccoons were trapped and tagged in Guthrie and Cerro Gordo counties during the spring, summer and fall of each year. Sex, age and weight were recorded for each animal and a blood sample was collected. Serum samples were tested for the presence of serum neutralizing antibodies (SNA) by the rapid fluorescent focus inhibition test (RFFIT), mouse serum neutralization test (MSN), and an indirect fluorescent antibody (IFA) technique for detecting immunoglobulin G. Fifty-one raccoons (5%) were found to have SNA by the RFFIT. Thirty-six serum samples (24 with RFFIT antibody titer >3.0, and 12 <3.0) were also tested by the MSN, with results correlating well with the RFFIT results (r = 0.86, P < 0.01, Kappa = 0.93). In 35 raccoons with SNA by the RFFIT, six individuals had immunoglobulin G binding activity by the IFA test. These results provided serologic evidence of exposure of raccoons to rabies virus in an area free of enzootic raccoon rabies.