Forty women participated in three clinic visits during which they were exposed to their partner's semen (10 μL, 100 μL, and 1 mL). At each visit they took vaginal fluid samples before exposure to their partner's semen, immediately after, and at 1, 24, and 48 h after exposure. PSA was measured with an enzyme-linked immunoassay. The mean PSA level for preexposure swabs ranged between 0.43 and 0.88 ng/mL. The mean PSA levels were 193 immediately after exposure to 10 μL, 472 after 100 μL, and 19,098 after 1 mL. The PSA levels declined within 1 h, and returned to background at 48 h. The findings confirm that our procedure is a sensitive and specific method for detecting recent semen exposure, and indicate that PSA levels depend on exposure intensity and time since exposure. Application of this method in condom efficacy studies provides objective evidence of condom failure that enhances the interpretation of self-report.