The Charity Commission has frozen the bank account of the Air Ambulance Foundation and opened a statutory inquiry.
According to its aims, the charity provides financial support to air ambulance services throughout England and Wales.
In a statement, the regulator said that the trustees failed to answer financial questions sent to the board.
The commission said the inquiry would examine whether trustees can account for how the charity spent its money and whether that spending supported its charitable objectives.
It may also extend the scope of the inquiry if additional issues emerge, it added.
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