The polymerase chain reaction (PCR) technique was compared with culture for the detection of Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma genitalium in clinical samples (vaginal secretions, throat and endotracheal secretions, and skin swabs) obtained from 47 high-risk pregnant women peripartum and eight newborn infants. Detection using PCR with homologous primers was highly specific, as a product with the expected length was consistently amplified with homologous but not with heterologous species. The limit of detection of the PCR assay was 10 color-changing units (CCU) of Mycoplasma strains. The PCR technique facilitated detection of Ureaplasma urealyticum DNA in 31 of 55 patients studied, of Mycoplasma hominis in seven samples, and of Mycoplasma genitalium in two samples. Four PCR-positive patients yielded culture-negative results. In one case a culture-positive sample was negative by PCR. The results show that PCR is a valuable tool for rapid detection of genital mycoplasmas in clinical samples. It is fast, sensitive, specific, and easy to perform, requiring minimal preparation of the clinical sample.