The case comes after claims by hundreds of women that their infants were taken from them at birth and given away in illegal adoptions.Doctors, nurses and religious workers at several clinics and hospitals in Spain are alleged to have sold babies for adoption over decades, after telling new mothers that their infants had died.
At the hearing, Maria Gomez Valbuena, a Sisters of Charity nun now in her 80s, became the first person accused in the widening scandal.
Dressed in a dark habit, she was questioned by a judge at Madrid's Superior Tribunal of Justice but invoked her right not to testify.
The formal charges against her are of illegal detention and falsifying documents in a case dating from the early 1980s.
Sister Maria Gomez Valbuena was called to give evidence in the case of Maria Luisa Torres, a mother who has accused the nun of stealing her daughter shortly after she was born at the Santa Cristina Hospital in Madrid in March 1982.
Ms Torres, 58, said that she accepted the nun's offer to temporarily look after her newborn daughter until her economic situation improved but that instead Sister Maria gave the baby away to another family.
Ms Torres was reunited with her daughter Pilar last year following an investigation by a journalist.
A crowd of mothers who say they were robbed of their babies shouted "shameless" at the nun as she was escorted out of court.
An association of parents and families, Anadir, has presented more than 900 lawsuits alleging child-stealing. However, most have been thrown out due to lack of evidence.
Many mothers say they were told by health or religious workers their babies had died at birth or shortly after, but were neither shown a body nor given a proper death certificate.
Anadir says the practice began in the 1940s when, in the aftermath of the Spanish Civil War, the fascist government stole babies from political prisoners from the defeated Republican side.
In subsequent decades it became a money-making racket, the victims claim.
Many of the mothers have said they believe their babies were taken due to a mistaken paternalism on the part of the doctors or religious workers, who may have seen them as unfit mothers because they were young, poor or unmarried.