OBJECTIVE: To determine the herpes simplex virus 2 (HSV-2) seroprevalence rate in a Melbourne antenatal cohort. DESIGN: Prospective collection of serum and questionnaires in 1371 women attending an outpatient antenatal clinic. SETTING: A tertiary obstetric hospital in metropolitan Melbourne. PARTICIPANTS: Women aged 18 years or older attending an antenatal clinic appointment. MAIN OUTCOME MEASURE: Seroprevalence rate of HSV-2 using an ELISA-based- type-specific serological assay. RESULTS: The overall HSV-2 seroprevalence rate in women was 13.6%. Only 0.4% of assays were equivocal and required confirmation by Western blot analysis. By multivariate analysis, HSV-2 seroprevalence was found to be associated with increasing age (odds ratio (OR) 4.63; confidence interval (CI) 1.86, 11.52 for age greater than 40 years), increasing number of sexual partners (OR 4.07, CI 2.13, 7.7 for five or more) and a past history of genital herpes in the index case (OR 5.48, CI 2.77, 10.87) or in a current or previous partner (OR 8.29, CI 4.15 to 16.56). CONCLUSIONS: HSV-2 seroprevalence rates in Melbourne are comparable to other similar populations in Australia. Routine antenatal screening for HSV-2 is probably not warranted but targeted screening based on numbers of sexual partners or a history of genital herpes in partners may be justified.