Polymerase chain reaction (PCR) was used to detect Mycoplasma (M) pneumoniae DNA in simulated clinical samples. Throat swabs were mixed with known amounts of broth-grown M. pneumoniae cells. An estimated detection limit of less than 40 colony forming units (cfu) was obtained without the need for time-consuming hybridization. The PCR is completed in one day and may be useful for the early detection of M. pneumoniae in clinical samples.