We compared polymerase chain reaction (PCR) analysis of specimens obtained from the distal vagina with wet mount microscopy and culture of specimens from the posterior vaginal fornix. One or all three techniques revealed that 61 (20.3%) of 300 women tested were positive for Trichomonas vaginalis. PCR analysis of distal vaginal specimens detected 56 (91.8%) of 61 infections, while wet mount microscopy and culture detected 49 (80.3%) of 61 infections. Results of this study may impact the approach to testing for T. vaginalis by eliminating the requirement of a vaginal speculum examination. The distal vagina is an appropriate testing site for T. vaginalis by PCR analysis.