AIM: To develop a sensitive and specific polymerase chain reaction for the detection of Pentatrichomonas hominis in biological specimens. METHODS: Three primers, associated in two primer pairs, were designed to amplify a sequence from the SSU rRNA gene of P. hominis. The specificity of both primer pairs was established by testing DNA extractions of different Trichomonad species, protozoa, bacteria, yeasts, and human leucocytes. The analytical sensitivity was determined through testing dilutions of P. hominis trophozoites. The clinical specificity and applicability of the assay was evaluated on stool samples and self-administered vaginal swabs. CONCLUSIONS: A highly specific and sensitive PCR assay was developed. Both primer pairs performed equally well. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of P. hominis in vaginal specimens has not been reported before.