The rapid diagnosis of intra-amniotic infection in patients with premature rupture of the membranes (PROM) and preterm labor is of utmost importance. The Gram's stain examination of amniotic fluid can only detect half of these infections. The acridine orange (AO) stain has been claimed to be superior to the Gram's stain in the detection of bacteria in biologic fluids. Therefore, we undertook the present study to examine the value of AO in the detection of bacteria in amniotic fluid. One hundred and thirty-eight patients with a culture-documented prevalence of intra-amniotic infection of 23.2% were studied. The diagnostic indices of the AO were: sensitivity, 43.8%, specificity, 97.2%, positive predictive value, 82.8%, and negative predictive value, 85.1%; the diagnostic indices for the Gram's stain were: sensitivity, 46.8%, specificity, 98.1%, positive predictive value, 88.2%, and negative predictive value, 85.9%. There was no difference in the diagnostic values of the tests. The agreement between the two techniques was substantial, as indicated by a kappa index of 0.72 (SE = 0.17, p < 0.001). The AO offered no significant advantage over the Gram's stain examination of amniotic fluid in the detection of intra-amniotic infection. However, the AO stain was able to identify mycoplasma infections that escaped detection by the Gram's stain. Therefore, the AO is presently the only microscopic technique capable of detecting mycoplasma in amniotic fluid.