Frei, president from 1964 to 1970, died of a sudden infection in January 1982 while he was hospitalized for hernia treatment. He was 71.
Pinochet became Chile's dictator soon after a military coup that killed Frei's successor, Salvador Allende, on September 11, 1973. Pinochet left office when democracy was restored in 1990.
Frei died when he and his Christian Democratic Party were gaining strength as the military regime's main opposition. The Pinochet regime faced its first street protests in the early 1980s.
A probe led by Judge Alejandro Madrid determined that dictatorship operatives killed Frei by poisoning. The new exhumation seeks to determine what type of poison was used.
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Two former agents of Pinochet's CNI political police face charges of murdering Frei, two doctors are accused of being accomplices, and two other doctors are accused of helping cover up the crime.
The suspects are currently out on bail.
Frei died in the same hospital where nine years earlier poet and Nobel laureate Pablo Neruda died. Chilean prosecutors believe that Neruda was poisoned by the Pinochet regime.