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Problem To determine the best method to detect semen in human vaginal secretions. Method of study Vaginal secretions from 302 pregnant women at mean 11.8 weeks’ gestation were analyzed. Semen detection was assessed with: (i) measurement of total prostate-specific antigen (PSA), (ii) acid phosphatase activity, (iii) microscopic measurement of spermatozoa on Gram stain, and (iv) self-reported sexual intercourse in the past 2 days. Sensitivity and specificity were calculated for each technique in comparison with PSA levels. Results A total of 119 (39.4%) women had a detectable PSA. Compared with measurable PSA, the sensitivity and specificity for other methods were: acid phosphatase (26.9%, 98.4%), Gram stain (36.1%, 98.4%), and self-report of intercourse in the past 48 hr (41.9%, 88.8%). Conclusion Compared with PSA levels, commonly used assays for recent semen exposure are inaccurate. This inaccuracy may affect the results of studies, which measure vaginal immune factors like cytokines or retrieve DNA from vaginal specimens.