On the basis of the nucleotide sequence of the multiple-banded (MB) antigen genes of Ureaplasma urealyticum, a polymerase chain reaction (PCR) technique was developed for rapid detection and biovar differentiation of U. urealyticum in a total of 100 urogenital specimens from 50 female patients. Positive PCR UM-1 amplification was found in 28 cervical swabs and 31 urine samples. Overall agreement between PCR and culture was 95%. Members of the two biovars of U. urealyticum could be distinguished by the size of the PCR UM-1 amplification products. Biovar differentiation was also demonstrated by two additional sets of PCRs: PCR UM-2 and UM-3. The PCR UM-2 was used to amplify biovar 1, while PCR UM-3 amplified biovar 2 specifically. The results indicated that use of the MB antigen gene as a target for PCR amplification could provide rapid and specific detection and biotyping of ureaplasma DNA in urogenital samples.