Recent reductions in numbers of human and equine Japanese encephalitis (JE) cases in Japan have seen calls to end JE vaccination. Here, we analyzed yearly variations of natural JE virus activity, using sera collected serially in 1998-2003 from racehorses residing in Ibaraki and Shiga prefectures, both located in central Japan. A total of 208 sera from 24 individuals in Ibaraki and 259 from 27 in Shiga were examined for antibodies to JE virus nonstructural 1 (NS1) protein, a marker of natural infection. The natural infection rate in epizootic seasons, which was determined by a significant increase in NS1 antibody level, was 4.2-26.7% in Ibaraki and 0-41.7% in Shiga, indicating that high levels of JE virus activity still existed in central Japan.