Objectives: Little information is available concerning PCR-based direct detection of Trichomonas infections on archived Pap (Papanicolaou)-stained smears. This study investigates DNA extraction and amplification from archived Pap smears. Trichomonas vaginalis is a parasitic protozoan that infects the urogenital tract of women. Study Design: DNA from archived Pap-stained smears was successfully amplified using the nested PCR to investigate if it could be used for accurate detection and retrospective epidemiological investigations. Results: In our study, 98 (75.4%) out of 130 specimens of T. vaginalis Pap-stained smears were found to be positive by the nested PCR. Also, direct PCR on the archived Pap smears for identifying T. vaginalis gave a specificity of 100%. Conclusion: PCR-based Pap smears appear to offer an effective method to detect Trichomonas infection in archived samples, being rapid, highly specific and convenient for sampling, particularly in retrospective investigations.