Timmons, an outside linebacker who is practicing at Joey Porter's old position, was reinjured during the first of a pair of two-hour practices and will be out indefinitely.
A few minutes before getting hurt, Timmons dropped into coverage during an offense vs. defense drill and intercepted a Charlie Batch pass in the flat.
"He made a couple of plays today, then he ducked out," coach Mike Tomlin said, joking. "He felt a little twinge, so we're being cautious. We don't want to take a step backward as we move forward, and we'll take a look at it and then go from there."
Timmons initially hurt himself May 11 and had not practiced again until this week, sitting out all the voluntary coaching sessions that lasted until early June. The Steelers have downplayed the importance of Timmons' absences - for now.
"Lawrence has got to make up some ground, it's not earth shattering, it's just the reality of it," Tomlin said. "He's got time to get better."
Timmons, who started one season at Florida State, downplayed the injury and wouldn't identify when it occurred, even though it was apparent when he was hurt during the 11-on-11 drill and trainers attended to him for the remainder of the morning practice.
"Nothing's wrong. It's no problem," Timmons said. "I don't think this will be a setback. Groins take a while to heal and you can't rush anything."
Timmons insisted he could play now if it was necessary.
"I could go, but the two-a-days (cause) wear and tear on your body, two hours each time, and that's hard on a groin when you've never done anything like that," he said.
On Sunday, Timmons reached terms on a four-year contract that guarantees him $8,053,000 in bonus money and could be worth as much as $15 million. Timmons is expected to press former backup James Harrison for playing time at right outside linebacker, Porter's position before he was released by Pittsburgh and signed with Miami in March.
Holmes caught 49 passes for 824 yards and two touchdowns as a rookie last season, starting four games. With new offensive coordinator Bruce Arians adding more multiple-receiver formations, Holmes is expected to take on a major role in the passing game this season.
"He's getting a little impatient, but that's part of it," Tomlin said. "He's a talented guy, a sharp guy, and he'll create opportunities for himself and catch up."