The purpose of this study was to determine the clinical significance of invasion of the amniotic fluid (AF) with Ureaplasma urealyticum in the midtrimester of pregnancy. Amniotic fluid and cervical swabs obtained from 214 asymptomatic women in the midtrimester of pregnancy (16-20 weeks) were cultured for U urealyticum and Mycoplasma hominis. Inoculum size was determined by quantitative culture. Six of the 214 women (2.8%) had Ureaplasma in high titers in the AF. The pregnancy outcomes of 129 women were determined. Adverse pregnancy outcome occurred more frequently in women with a positive AF culture than in women with a negative AF culture (3/6 [50%] vs. 15/123 [12%], respectively; P = .035). We conclude that infection of the amniotic cavity with U urealyticum can be present in asymptomatic patients in the midtrimester of pregnancy and is a significant risk factor for spontaneous preterm labor and delivery. These observations suggest that routine culture of AF at the time of midtrimester amniocentesis can identify the group of patients at risk for a poor pregnancy outcome.