On the basis of the nucleotide sequence of the urease genes of Ureaplasma urealyticum serotype 8, polymerase chain reaction (PCR) primers were selected, evaluated for specificity and sensitivity, and tested for their ability to detect U. urealyticum in the adult urogenital tract, amniotic fluid, and endotracheal aspirates of newborns. All 14 reference serotypes of U. urealyticum were detected with equal sensitivity (1-10 cfu), whereas multiple strains of 12 other mycoplasma species found in humans as well as eukaryotic DNA were not detected. A total of 638 clinical specimens was evaluated. Results indicate that PCR is equal to if not more sensitive than culture for detection of U. urealyticum. Faster detection of U. urealyticum by PCR (< 24 h) compared to culture (2-5 days) will be particularly important in management of very low birth-weight infants in whom this organism has been shown to be a significant cause of meningitis, respiratory disease, and death. This method of detection will also be helpful in further determining the role of this organism in intraamniotic infection and premature birth.