Australian Kyrgios, who has beaten Novak Djokovic twice and taken Roger Federer close over the last month on the back of some brilliant serving, is tied for third place with the tall American on the ATP leaderboard with 218 aces this season.
The clash between world number 16 Kyrgios and the 23rd ranked Isner will follow Jack Sock's first meeting with 22-year-old Australian Jordan Thompson on the hardcourt of the Pat Rafter Arena.
"I'm looking forward to getting back here and having some home support," said Kyrgios, who had to deal with a hostile crowd when he lost to Federer in Miami last week.
"I feel comfortable playing second. I'll be out there for some of Thommo's match and I think it's a good match up with him against Sock."
Another Australian big server, Sam Groth, will team up with John Peers to take on Sam Querrey, who is no slouch in the service department, and Steve Johnson in Saturday's doubles before the reverse singles on Sunday.
