An Iranian-flagged ship carrying humanitarian relief to Yemen should instead deliver the cargo to neighboring country Djibouti, the Pentagon said.
White House Press Secretary Josh Earnest told reporters the United States is monitoring the ship, which is heading toward the Strait of Hormuz between Oman and Yemen.
He said Iran should be following United Nations protocols for delivering aid to Yemen by dropping it off in Djibouti.
"Iran understands that they can't afford to play games with humanitarian assistance to people who are in dire need," he said. "The Iranians know as well as anyone that a political stunt to defy their regional rivals outside the UN system is provocative and risks a collapse of the UN-led humanitarian cease-fire."
Pentagon spokesman Col. Steven Warren told reporters Tuesday that if Iran is "planning some sort of stunt," it would be "unhelpful."
One US defense official told CNN the ship doesn't appear to be carrying any weapons but there are some concerns that Iranian warships will attempt to escort the vessel to Yemen. Warren said that was "absolutely unnecessary."
Iran's humanitarian aid ship is several days from arriving in Yemen.
The Strait of Hormuz has been the site of simmering tensions between the United States and Iran after Iranian Revolutionary Guard ships harassed the US-flagged Maersk Kensington commercial vessel April 24, then seized the Marshall Islands-flagged Maersk Tigris on April 28.
The US Navy began accompanying all US-flagged commercial ships through the strait over safety concerns, but stopped doing so last week.
Meanwhile, a United Nations-led ceasefire went into effect in Yemen on Tuesday just hours after airstrikes pounded rebel Houthi positions in Sanaa and Aden.
Fighting intensified between the Yemeni military, aided by air support from a ten-nation coalition of Arab countries, and the Iran-supported Houthis, who overtook the government in late 2014. Both sides agreed to a five-day humanitarian ceasefire.
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