Rickettsial infection of cattle and sika deer from a pastureland in Hidaka District, Hokkaido, Japan was examined by serological and molecular methods. Serum samples from 8 of 83 (9.6%) cattle reacted with Rickettsia helvetica antigens in an IFA test, with titers ranging from 1:160 to 1:640, while serum samples from 15 of 22 (68.2%) deer were positive for R. helvetica, with titers ranging from 1:80 to 1:640. In a genus-specific nested PCR based on gltA, no cattle were positive for Rickettsia, while 14 of 22 (63.3%) samples obtained from deer tested positive. Sequence analysis revealed that positive samples from sika deer showed 100% nucleotide sequence identity with the known sequence of Rickettsia asiatica.