The basement was searched in 1979, the year the boy disappeared, but the floor was never dug up.
Since then, drywall has been put up over the room's brick walls. The drywall will be removed and the bricks examined and tested for blood evidence using advanced forensic techniques that were not available three decades ago, officials said.
The floor will also be dug up in a search for human remains, clothing or other evidence.
For the Patz family, it has been more than three decades of agonizing investigations and years of wondering what happened to their blond son with the gorgeous smile.
In an interview with "20/20" in 2009, the boy's father Stan Patz said, "I still gag with fear that this child must have felt ... when he realized he was being betrayed by an adult."
The case had been dormant until Manhattan DA Cy Vance Jr. reopened the case. Former DA Robert Morgenthau had declined to proceed with the case, citing insufficient evidence.