The Serious Fraud Office has launched an investigation into payments made between Barclays and Middle East investors it tapped for funds at the height of the financial crisis.
The inquiry, which comes after a similar probe by the FSA was revealed last month, is a fresh blow to the bank which is still reeling following the Libor rate-rigging scandal.
Barclays raised billions of pounds from Middle East investors in 2008, which effectively allowed it to avoid following in the footsteps of Lloyds and Royal Bank of Scotland by taking a state bailout.
The SFO is looking in particular at payments between the bank and Qatar Holding - part of sovereign wealth fund Qatar Investment Authority - which is understood to have made a £2 billion investment.
Whereas the FSA's investigation was centred on four present and past senior staff, including finance director Chris Lucas, the SFO's probe is currently not thought to be focusing on any individuals.
Barclays raised more than £10 billion in emergency funding in two fundraisings in 2008.
The bulk of this came from the Middle East, including a £3.5 billion investment from was from Manchester City owner Sheikh Mansour Bin Zayed Al Nahyan - a member of Abu Dhabi's royal family - which is not part of the investigation.
The SFO's move will ramp up pressure on Barclays after it already opened a criminal investigation into the the Libor rate-rigging scandal.
Barclays has endured one of the most turbulent periods in its history after it was fined £290 million by UK and US regulators for manipulating Libor, an interbank lending rate which affects mortgages and loans.
It is the only bank to have admitted attempting to rig Libor, though several others are also the subject of international investigations. The affair led to the departure of chief executive Bob Diamond, chairman Marcus Agius and chief operating officer Jerry del Missier.