Background and Objectives: Sexual transmission of bacterial and viral sexually transmitted disease has been reported between women. No data are available on seroprevalence of herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) among lesbians. Goal: The goal was to define prevalence of infection with HSV-1 and HSV-2 among lesbians, and associated risk factors. Study Design: Women who reported sex with another woman in the preceding year were eligible. Medical and sexual histories were obtained. Serum was tested for HSV-1 and HSV-2 antibodies using Western Blot assay. Results: Among 392 subjects, antibodies to HSV-1 were detected in 46% and to HSV-2 in 8%. Increasing age predicted higher seroprevalence to both HSV types, and HSV-2 seropositivity was associated with a history of male partner with genital herpes. Of 78 women reporting no prior sex with men, 3% were HSV-2-seropositive. Most HSV-2-seropositive subjects (71%) reported no history of genital herpes. HSV-1 seroprevalence increased significantly with an increasing number of female sex partners. Conclusions: HSV-2 infection occurs in nearly 1 in 10 lesbians and is not predicted by report of sex with men or sexual identity. Most lesbians infected with HSV-2 are not aware of their infection. Sexual transmission of HSV-1 may occur more frequently among lesbians than among heterosexual women.