The association between the detection of Mycoplasma hominis or Ureaplasma urealyticum in midtrimester amniotic fluid and amniotic fluid cytokine concentrations and subsequent pregnancy outcome were examined. Amniocentesis was performed between 15 and 19 weeks of gestation in 179 asymptomatic women. Aliquots were assayed for M hominis and U urealyticum by polymerase chain reaction coupled to enzyme linked immunosorbent assay. Intra amniotic levels of interleukin- 1β , interleukin- 1 receptor antagonist, interleukin- 4, interleukin- 6, and tumor necrosis factor- α were determined by enzyme linked immunosorbent assay. Pregnancy outcomes were obtained after the completion of all testing. U urealyticum was detected in 22 of 172 amniotic fluids (12.8% ); M hominis was present in 11 of 179 amniotic fluids (6.1% ). There was no relationship between U urealyticum detection and the concentration of any cytokine. Detection of M hominis was associated with elevated intra amniotic concentrations of interleukin- 4 (P =. 01). Preterm premature rupture of membranes that was followed by preterm birth occurred in 5 women (2.8% ); 5 women (2.8% )- had a spontaneous preterm birth with intact membranes. All 5 of the women with preterm premature rupture of membranes (100% ) tested positive for either U urealyticum or M hominis, as opposed to none of the women with spontaneous preterm birth and to 27 of 161 women (16.8% ) with a term birth (P =. 0002). The detection of M hominis or U urealyticum in midtrimester amniotic fluid by polymerase chain reaction enzyme linked immunosorbent assay may be a risk factor for subsequent preterm premature rupture of membranes. The association between the detection of Mycoplasma hominis or Ureaplasma urealyticum in midtrimester amniotic fluid and amniotic fluid cytokine concentrations and subsequent pregnancy outcome were examined. Amniocentesis was performed between 15 and 19 weeks of gestation in 179 asymptomatic women. Aliquots were assayed for M hominis and U urealyticum by polymerase chain reaction coupled to enzyme linked immunosorbent assay. Intra amniotic levels of interleukin-1β, interleukin-1 receptor antagonist, interleukin-4, interleukin- 6, and tumor necrosis factor- α were determined by enzyme linked immunosorbent assay. Pregnancy outcomes Urealyticum was detected in 22 of 172 amniotic fluids (12.8%); M hominis was present in 11 of 179 amniotic fluids (6.1%). There was no relationship between U urealyticum detection and the concentration of any cytokine. Detection of M hominis was associated with elevated intra amniotic concent rations of interleukin-4 (P = .01). Preterm premature rupture of membranes that was followed by pre-term birth with intact in 5 women (2.8%); 5 women (2.8%) - had a spontaneous preterm birth with intact membranes. of the women with preterm premature rupture of membranes (100%) tested positive for either U urealyticum or M hominis, as opposed to none of the women with spontaneous preterm birth and to 27 of 161 women (16.8%) with a term birth (P =. 0002). The detection of M hominis or U urealyticum in midtrimester amniotic fluid by polymerase chain reaction enzyme linked immunosorbent assay may be a risk factor for subsequent preterm premature rupture of membranes.