Polymerase chain reaction (PCR)-based DNA testing of archival cervical smear slides is a useful method of retrospectively establishing the presence of the human papillomavirus (HPV) in cervical cells. A cellular DNA recovery test is performed in parallel to HPV DNA testing to ensure that sufficient cells are present and purification of sample DNA has been successfully performed. Previous studies have not comprehensively assessed DNA recovery rates in slides older than 13 years. We undertook a study to determine the factors impacting DNA recovery in 436 UK slides dating from 11 to 33 years prior to testing. Overall, a low cellular DNA recovery success rate of 29% was obtained but a strong trend was observed with increasing recovery rates the older the slides (P < 0.001). Recovery rates increased from 22% in the most recent slides collected from 1988 to 1992, to 61% in the oldest slides, collected in 1970-72. It is likely that fixation compounds incorporating acetic acid, introduced in the UK through the 1980s, have compromised subsequent attempts at PCR amplification. These findings emphasize the importance of the original fixation method in the success of DNA recovery from archival smear samples.