Background: Ureaplasma urealyticum has been implicated in chronic lung disease in very low birth weight (VLBW) infants, although its role remains controversial. The “gold standard” for diagnosing Ureaplasma pneumonia is by culture of endotracheal aspirate fluid, which may take 3 to 7 days and thereby unnecessarily delay therapy. Polymerase Chain Reaction (PCR), a method by which a specific DNA sequence is amplified, has been applied as a rapid diagnostic tool for a variety of infectious agents. We hypothesized that PCR amplification would be a more rapid and sensitive method for the diagnosis of Ureaplasma urealyticum pneumonia in the VLBW infant and surveyed our population to determine the incidence of this organism. Method: We performed PCR of the urease structural gene on inoculated culture media and/or endotracheal aspirates obtained from 99 intubated VLBW infants. Results: PCR was 100% sensitive when compared to culture, with additional patients being identified by PCR who had negative cultures. The incidence of Ureaplasma in our intubated VLBW population was 28% by culture and 35% by PCR. Conclusion: PCR of the urease gene is both rapid and reliable, and is useful in identification of Ureaplasma. Due to its superior ability to detect the organism, PCR should become the method applied in clinical trials to determine pathogenic effects of Ureaplasma. Continued use of culture in such studies allows those patients who are culture-negative, but PCR-positive, to be included in control groups which may be contributing to further confusion about the pathogenicity of Ureaplasma. Our patients have a substantially higher incidence of Ureaplasma than has been published previously. This suggests that the rate of colonization may vary based on geographic constraints, and that incidence of the organism in a population should also be considered in clinical trial design. Finally, using PCR as the method of choice for early diagnosis and therapy could reduce chronic lung disease due to Ureaplasma in the VLBW infants.