Gregory Villemin was found, his hands and feet tied, drowned in the cold waters of the Vologne river in eastern France in October 1984, sparking a long and convoluted legal saga that transfixed France for years.
The death of "Little Gregory", as he became known, led to one of France's most notorious post-war murder mysteries, as police sought to untangle a web of family hatred and local jealousies.
On Wednesday morning, nearly 33 years after the murder, police arrested Marcel Jacob, an uncle of Gregory's father, and Jacob's wife in the Vosges mountains.
Ginette Villemin, half-sister to Gregory's father Jean-Marie Villemin, was also detained in the same region, police sources told AFP.
Gregory's grandparents were also questioned but were later released.
The arrests "target people very close to the heart of this case and aim to clarify certain points and to provide answers to questions we have," local prosecutor Jean-Jacques Bosc said in a statement.
"This is a giant step on the path to the truth," said Thierry Moser, the lawyer acting for the dead boy's parents.
The case was reopened in 2010 to take advantage of new DNA techniques.
The day after Gregory's body was found, a letter arrived at the home of the child's parents claiming responsibility for the murder and calling it "revenge". The couple had been receiving anonymous hate mail since 1981.
Investigators took that letter seriously because it seemed to have been posted before the body was found.
Handwriting experts identified Jacob -- the uncle of Gregory's father who was among those held on Wednesday -- as possibly being the writer of the letter.
Bernard Laroche, a cousin of the child's father, was charged with the murder a month after the boy's death, based on evidence given by a teenage sister-in-law.
He was released after she withdrew her claims, only to be shot dead in March 1985 by Gregory's father Jean-Marie Villemin who spent two and a half years in prison for the crime.
The dead boy's mother, Christine Villemin, was herself charged with the murder in 1985. But she was finally cleared eight years later and all charges against her were dropped.
The arrests on Wednesday were on charges of being an accomplice to murder, failing to denounce a crime and failing to help someone in danger, said local newspaper l'Est Republicain, which broke the story.
In the course of this latest probe into the case, investigators questioned around a hundred witnesses, some for the first time, prosecutor Bosc said in his statement.
They also analysed more than 2,000 anonymous letters received by those involved in the affair, including some to magistrates working on the case.