Two hundred seventy-nine serum samples from men attending sexually transmitted disease (STD) clinics in Baltimore, Maryland, were tested for herpes simplex virus type 2 (HSV-2)-specific antibody by three immunosorbent glycoprotein G-2-based assays (the Kalon, Focus, and Biokit assays). The results for all samples with positive results were confirmed by Western blotting (91/279; 32.6% HSV-2 seroprevalence). All patients were also tested for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, human immunodeficiency virus type 1, and hepatitis C virus. The Kalon assay performed very well with samples from this population (90.8% sensitive, 99.4% specific), whereas the Focus assay had a sensitivity (82.6%) much lower than that shown previously. For 19.7% of the samples, the Biokit assay gave an indeterminate result. It was found that the odds of a sample having a Biokit assay indeterminate result compared to that of having a definitive positive or negative results were 3.88 times greater for subjects concurrently infected with N. gonorrhoeae, after the effects of other STDs were controlled for (P = 0.001; 95% confidence interval, 1.78, 8.45). Unfortunately, we were unable to control for HSV-1 infection status in the regression model, which, on the basis of chi(2) analysis, might also affect the clarity of the Biokit test. The recommended index cutoff value of 1.1 for the Focus and Kalon assays was found to be optimal for this population.