The response follows press reports about ambulance service "turnaround times", which appear to show thousands of patients wait at least an hour before being admitted.
But a DH spokeswoman said allegations of so-called "patient stacking" were "completely untrue".
"The four-hour waiting-time target starts 15 minutes after an ambulance arrives, regardless of whether the patient is in the ambulance or the hospital," she said.
London Ambulance Service trust - one of seven services to provide figures - showed 14,700 turnaround times of an hour or more for last year.
But a spokesman said these were not a fair way to gauge handover times. "The average turnaround time last year - from arrival at hospital to a crew becoming available to attend another call - was 33 minutes," he said. "For cases above this average, there is no automatic link to the time it may take for a patient to be handed over into the care of hospital staff."
For example, a serious assault victim would be seen immediately but it might then take longer to clean the ambulance and staff could have to spend time talking to police and managers, he explained.
North West Ambulance Service trust recorded 6,643 turnarounds of an hour or more. A spokeswoman said: "We are the biggest ambulance service so [the figures] only represent 1-2 per cent of turnaround times."
However, some services admitted there had been problems with patients waiting in ambulances. A spokesman for West Midlands Ambulance Service trust, which reported 9,376 turnarounds of an hour or more, said some patients had waited up to two hours.
One reason was that it is hard to predict spikes in demand, he said. But he stressed that seriously ill patients would always be seen straight away. "We have put in place measures, which the strategic health authority backs and acute trusts have taken on board, that mean we're addressing that issue and the numbers are down," he said.
The DH has hailed progress on accident and emergency waiting times as a major victory. National emergency access director Sir George Alberti said: "Four years ago, almost a quarter of patients spent more than four hours in A&E; now over 98 per cent are seen, diagnosed and treated within four hours of their arrival."
The patient stacking claims followed the latest waiting-time figures for emergency units, which show that from October to December 2007, just over 123,000 patients waited more than four hours. That is up 55 per cent from the previous quarter and up just over half on the same period the previous year.