Television images showed parts of SpaceShipTwo, a test vehicle flying to the edge of space, scattered amid brush in the desert east of Mojave, a few hours' drive northeast of Los Angeles.
The incident is second disaster involving a US spacecraft this week, after an unmanned Orbital Science rocket exploded on Tuesday six seconds after launch on a resupply mission to the ISS.
"During the test, the vehicle suffered a serious anomaly resulting in the loss of SpaceShipTwo. Our first concern is the status of the pilots, which is unknown at this time," the firm said in a tweet.
The spaceship had been carried aloft on a bigger aircraft known as WhiteKnightTwo and then released for a test of its rocket engine above the Mojave desert, in what was the 35th such flight.
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"SpaceShipTwo has been released by WhiteKnightTwo, and is now flying freely," the firm wrote in a blow-by-blow account of the flight, adding: "Ignition! SpaceShipTwo is flying under rocket power again."
The next tweet announced the "anomaly", followed by the series confirming the crash. The company added that WhiteKnightTwo had landed safely after the accident.