Ryanair has accused lastminute.com and similar online travel traders of "ripping off" consumers by adding up to 133% to the cost of its flights.
But the online sites, which have become popular with the travelling public, insist customers are getting a good deal.
The Dublin-based economy airline claims its flights are being sold by "middle men" at "inflated prices" when consumers can buy direct from it at a fraction of the cost.
Its investigation into fares offered by tour operators found that flights were being sold on at premium rates.
The airline also claims some tour operators provide misleading information on baggage allowances, fail to advise passengers of flight changes, deny passengers the chance to pre-book bags online and do not provide web check-in and priority boarding options.
Ryanair says it now plans to block future business with online sites, although it accepts it may be difficult to stop tickets becoming available through independent travel websites.
According to the airline, one Dublin to London trip was being sold by lastminute.com for 45.18 euros when the Ryanair online price was half of that.
It said a flight from London to Dublin was being offered by another online company for £34.99 when the equivalent Ryanair price was £14.99.
Other flights were being sold on by middlemen to consumers with a mark-up of between 100 and 109%.
Ryanair's head of communications, Peter Sherrard, said that it would do all it could to block business from the likes of lastminute.com but admitted it could not stop third party traders from buying its flights to sell on to customers.
"The main point is that people think they are getting good deals from these online travel agents but it is just not true.
"Ryanair sells directly to the public, so passengers should avoid tour operator websites which charge extortionate mark-ups," he said.
"You might wonder why Ryanair is doing this because it still gets the passenger numbers from these sites. But there are issues about customers not being advised on important information, such as schedule changes, by these companies which do create genuine difficulties."
The demand for package tours has declined from 2002 when it accounted for 52% of all holidays to 44% in 2006, with analysts partly attributing this to the increased popularity of internet-based companies such as lastminute.com which allow customers to plan and organise their own custom-made holidays online.
John Bevan, travel director at lastminute.com, insisted its customers were getting a "competitive deal" with Ryanair flights.
"We can confirm that Ryanair is fully aware and satisfied with the terms and conditions published on our site, and we've been working with them for the past two years.We have not been blocked by them," he said.
"Flight-only sales from low-cost carriers such as Ryanair contribute to a small percentage of overall bookings. Self-packaging a flight with a hotel is where our customers can benefit from major savings as the fee can be offset by the massive hotel discount we negotiate for our clients.
"We would like to reassure our customers that when they book with lastminute.com they will always get a competitive deal.
"In line with other agents, when customers buy Ryanair flights a booking fee is added to the airline's fare.
"We offer customers a first-class service, which includes notifying them of any schedule changes to booking."
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