Women with invasive carcinoma of the cervix were examined for evidence of previous infection with herpes simplex virus (HSV) type 2 (HSV-2) by three assays: Assay 1 measured the ratio of lymphocyte blastogenesis to herpes simplex virus type 1 (HSV-1) and HSV-2 antigens; assay 2 measured the ratio of antibody titer to surface antigens of HSV-1-infected and HSV-2infected cells; assay 3 measured the titer of type-specific antibody to HSV-1 and HSV-2, including the percentage of HSV-2 type-specific to total HSV antibody. The ability of assays to detect previous infection was evaluated with the use of groups with histories of infection with HSV-1, HSV-2, or both. Women with invasive cervical cancer were matched to control women by age (±3 yr), race (black), and socioeconomic class (low). All assays detected similar patterns of previous HSV-2 infection, and all indicated a higher frequency of previous HSV-2 infection in women having cervical cancer. Results from assays 1 and 2 suggest that many of the cancer patients and controls also had been infected with HSV-1. The more definitive type-specific antibody assay confirmed those results and showed that sera from 85% of women having cervical cancer and from 100% of the matched controls contained antibody type-specific for HSV-1.