Between May 1, and September 15, 1964, neutralizing antibody to Powassan virus was detected in sera from 163 of 464 forest mammals captured in the Powassan-North Bay area of northern Ontario. These included 159 of 358 groundhogs and four of 43 red squirrels. Acquisition of antibody by juvenile groundhogs occurred principally during July and August. Powassan virus strains were isolated from tick pools containing two to 15 Ixodes cookei per pool which were removed from eight of 91 groundhogs in three townships during May, July and August. Virus was also recovered from blood of two groundhogs during May. Powassan virus was re-isolated from five of six tick pools and two blood clots by inoculation of swine kidney tissue cultures. These findings strongly suggest that during 1964 Powassan virus was maintained in nature by a cycle involving groundhogs and I. cookei ticks.