The Lyme disease spirochete Borrelia burgdorferi was detected in the adult stage of the ixodid ticks Ixodes persulcatus and I. ovatus collected in various localities in Japan. The total prevalence values were 16.6% of 777 I. persulcatus and 23.6% of 687 I. ovatus. Sixty-two (63.9%) of 97 isolates from I. persulcatus contained the 30-34-kD heterogeneous outer surface protein A (OspA) that reacted with monoclonal antibody H5332. In contrast, all 128 isolates from I. ovatus were homogeneous with regard to the type of 30-kD OspA protein, and were different from the I. persulcatus isolates in their protein composition. These data suggest that the transmission cycle of B. burgdorferi in I. ovatus is not related to that in I. persulcatus. No cases of Lyme disease transmitted by I. ovatus have been confirmed in Japan, in spite of the abundance of human tick bites by this species. Accordingly, the spirochetes of I. ovatus are thought to be a variant of B. burgdorferi with low virulence.