BACKGROUND: Most genital herpes simplex virus type 2 (HSV-2) infections are unrecognized, thus, strategies to reduce the sexual transmission of HSV-2 are partly dependent on serologic screening. GOAL: To define performance characteristics of the Gull/ Meridian glycoprotein G-based HSV-2 enzyme-linked immunosorbent assay among sexually transmitted disease clinic attendees and correlates of test acceptance. STUDY DESIGN: The cross-sectional study was conducted during two periods. Serologic testing was offered at a US $15 charge during the first period and at no charge during the second period. Sera were tested by a type-specific glycoprotein G enzyme-linked immunosorbent assay and Western blot analysis, with the latter test used as the reference standard. RESULTS: Acceptance of HSV-2 testing was associated with free testing (odds ratio, 7.5; 95% CI, 6.0-9.9), older age, and white race. Sensitivity of the HSV-2 assay was 80.5% and specificity was 98.5%. The HSV-2 positive and negative predictive values were 95.8% (95% CI, 91.6-98.0%) and 92.2% (95 % CI, 89.6 -94.2%), respectively. Antibodies to HSV-2 were detected in 25.9% of 606 persons with no history of genital herpes. CONCLUSION: Acceptance of HSV-2 serologic testing was cost sensitive. In this high-prevalence population, the positive predictive value of the enzyme-linked immunosorbent assay was sufficient to warrant its use without a confirmatory test. This assay could be useful in the screening of sexually active adults to detect unrecognized HSV-2 infection.