Speaking softly and politely, she testified that the boys who spent the night had a choice of where to sleep in her home, not just the basement. She told the jury her husband would go down and tell them goodnight, and that she made frequent trips to the basement while the boys were there and never noticed anything unusual.
Dottie Sandusky also said the basement wasn't soundproof, contradicting one accuser's testimony that he screamed during an assault but couldn't be heard. When asked about the accusers, she said some of them had troubling behavior, describing one boy as demanding and conniving and another as very "clingy" who would occasionally jump on Sandusky.
She also said she and her husband had always shared a bed and that her husband would usually go to bed first.
Defense lawyers called the former Penn State assistant football coach's wife to the witness stand after they went after two investigators, suggesting that police may have planted seeds of the sexual abuse accusations in Sandusky's accusers.
They also said investigators encouraged the accusers to confess to the alleged abuse by telling them other victims had already given detailed accounts of their encounters with Jerry Sandusky.
Their arguments were built upon stringing together evolving testimony from accusers to details investigators shared with them in interviews.
In one instance, a retired state police corporal initially said that he did not recall sharing details of specific sex acts with accusers. Then, defense attorney Joe Amendola read him an interview transcript where he told one accuser that police had received reports of oral sex and rape involving Sandusky.
A psychologist, Elliot Atkins, also took the stand Tuesday. He told jurors that he diagnosed Sandusky, 68, with histrionic personality disorder after talking with the ex-coach for six hours. People with the disorder often interact with others in inappropriately seductive ways, Atkins explained.
Meanwhile, NBC aired a previously unreleased portion of an interview with Sandusky from last November, which could be used against him.
"I didn't go around seeking out every young person for sexual needs that I've helped," Sandusky said in the interview. "There are many that I didn't have - I hardly had any contact with who I have helped in many, many ways."
All eyes were on Sandusky as the possibility of his testimony loomed. Sandusky is facing 51 child sex-abuse charges.
Sandusky did not reveal much when he arrived at the courthouse Tuesday, but his defense attorney did tell one reporter to "stay tuned" when she asked if he would be taking the stand.
The defense is expected to finish its testimony Wednesday and closing statements could come Thursday morning.
The prosecution on Monday wrapped up their case with testimony from the mother of one of the alleged victims. She said Sandusky gave her son clothes and other gifts, and that he always appeared to have stomach problems during his visits with Sandusky.
The defense then began their case with several character witnesses, one who called Sandusky "a local hero." Another said showering with boys was not unusual in the athletic world. But it was a point the prosecution could not understand.
"There was this odd and bizarre attempt to convince the jury somehow that showering [with young boys] is culturally accepted in this world. I don't see how that takes anyone very far," said Thomas Klein, attorney for the alleged victim known as Victim 5.
ABC News and the Associated Press contributed to this report.