The diagnosis of Mycoplasma pneumoniae infection currently relies on serological methods which may be slow to produce diagnostic results and may be inconvenient for both the clinician and the patient. This study was designed to assess whether or not the polymerase chain reaction (PCR) is a useful additional diagnostic method. Comparison was therefore made with serology as it is routinely practiced. PCR was used to examine for the presence of M. pneumoniae DNA in throat swab specimens obtained from 99 hospitalized patients investigated for a range of respiratory pathogens including M. pneumoniae. PCR detected M. pneumoniae DNA in 24 adults and 25 children, which is significantly more than the 32 patients found to be antibody positive by the particle agglutination test (p = 0.001). M. pneumoniae DNA was not detected in any of the throat swabs from 32 apparently healthy volunteers. PCR inhibitors were not detected in any of the samples tested. Significantly more children (88%) than adults (38%) were found to be anti-mycoplasma antibody-positive (p < 0.0001). Routine clinical practice was reflected in the fact that 56 patients (57%) had indeterminate serological results because only single sera were obtained. The sensitivity and specificity of PCR were assessed to be 92% and 98% respectively, using a combination of serological and clinical data as the benchmark. PCR appears to have advantages over serological testing, both with respect to accuracy and convenience of single specimen testing. The poor performance of serological tests in adults makes PCR especially useful in this age group.