Grealish is under a club investigation after reports police were called to a Birmingham hotel on Sunday morning, where the 21-year-old was alleged to be partying.
A newspaper report claimed fellow party-goers were inhaling nitrous oxide. There is no suggestion Grealish joined them doing so.
Villa have not finished their probe and Grealish - who switched allegiance to England, having played with Ireland U21s - has not been fined or disciplined yet, although both options remain open to the club.
Villa host Newcastle in the Sky Bet Championship tomorrow and Di Matteo refused to say whether Grealish, who signed a new four-year deal this month, will be dropped as he revealed his disappointment.
He said: “It’s still on-going, I cannot confirm or deny if he will be available. It’s a timing issue and we want to get it right.
“In general for the sake of any player they need to learn about what it takes to be an athlete. He needs to grow up very quickly.
“It’s not the first time and he needs to understand if he wants a good career he has to stop.”
Di Matteo is the fourth Villa manager to take academy graduate Grealish to task over his behaviour after Paul Lambert, Tim Sherwood and Remi Garde.
Sherwood was forced to answer questions about the England U21 international’s conduct after he was pictured passed out in the street on holiday in Tenerife in 2015.
Frenchman Garde also dropped Grealish last season after he partied in Manchester following a 4-0 defeat to Everton in November.
Di Matteo added: “First it’s a feeling of disappointment. I expect all players to behave in a professional manner at all times.
“We instantly started an investigation which is still on-going.
“Once it’s finished and we have all the facts then we will take disciplinary action.
“This is the stance.”