Karlovic, a former top-15 player now ranked 63rd, had surgery on his left Achilles tendon and hasn't played since the Madrid Masters in May. The U.S. Open is the third consecutive Grand Slam tournament he'll miss after entering 26 in a row.
Ancic has a back injury and will be skipping a seventh straight major championship. He has been ranked as high as No. 7 and is currently 469th, but was given a spot in the U.S. Open based on a protected ranking of 65th. He last played on tour in May at Munich.
The year's last Grand Slam tournament starts Aug. 30.