Objectives: Although genital ulcer disease (GUD) has been associated with human immunodeficiency virus (HIV) infection in a number of studies, definitions of genital ulceration have varied. The authors hypothesized that the association of GUD with prevalent HIV infection may vary according to the definition of GUD that is used. Methods: As part of a prospective cohort study, 863 patients were interviewed and examined who presented to a sexually transmitted disease (STD) clinic for new symptom evaluation and who agreed to HIV testing to determine demographic and behavioral risk associated with prevalent HIV infection. To determine the association between GUD and prevalent HIV, the following definitions of GUD were used: observed ulcers history of syphilis serologic evidence of syphilis, observed culture-proven genital herpes, and serologic evidence of herpes simplex virus type II (HSV- 2) infection. Results: Of 481 men and 382 women enrolled, prevalent HIV infection was detected in 12.5% and 5.2%, respectively. In multivariate analyses controlling for known HIV risk behaviors, prevalent HIV infection was associated with observed GUD (odds ratio [OR] = 2.0, 95% confidence intervals (CI) = 1.0-3.9), a history of syphilis (OR = 6.0, CI = 2.8-12.7), and serologic evidence of syphilis (OR = 3.7, CI = 1.9-7.0), but not with serologic evidence of HSV-2 (OR = 1.2, CI = 0.7-2.1), nor with observed HSV- 2 culture-positive genital ulcerations (OR = 1.0, CI = 0.4-4.2). Factors contributing to different strengths of association between HIV refection and a history of syphilis or serologic evidence of syphilis included the presence of under diagnosed syphilis infection in people with reactive serologic tests and the absence of serologic reactivity in people with a positive history. Conclusions: Although GUD is strongly associated with prevalent HIV, the strength of the association depends on the definition of GUD used. For accurate evaluation of people at risk for HIV, clinicians and researchers should use multiple definitions of GUD.