Polymerase chain reaction (PCR) amplification of the urease genes of Ureaplasma urealyticum was compared with culture for detection of the organism in 100 endotracheal aspirates from 54 ventilated preterm infants. Ninety specimens gave negative results by both culture and PCR and three specimens gave positive results by both culture and PCR. Six specimens were negative by culture but positive by PCR. The one specimen positive by culture and negative by PCR was interpreted as a false-positive culture result. Overall agreement between results obtained by culture and PCR was 93%. PCR is a sensitive and reliable method for the detection of U. urealyticum in neonatal endotracheal secretions. Detection by PCR (1-2 days) is more rapid than culture (2-5 days) and this will be important if early therapeutic intervention is shown to be effective.