A case-control study of 766 histologically confirmed incident cases of invasive cervical cancer and 1,532 hospital and community controls was conducted in Latin America to evaluate the etiologic role of herpes simplex virus type 2 (HSV-2) and to examine whether HSV-2 interacts with other risk factors. In addition to a personal interview, all subjects were asked to donate blood samples and cervical swabs for assessment of exposure to HSV-2 and human papillomaviruses (HPVs) respectively. Ninety-eight percent of cases and 91% of controls agreed to the interview and blood collection. Women testing positive for HSV-2 antibodies were found to have a 60% increased risk of cervical cancer compared with seronegative women (95% CI = 1.3, 1.9). Control for education, sexual and reproductive behavior, prior Pap-smear screening, smoking, oral contraceptive use, HPV-6/11 DNA, or HPV-16/18 DNA detection did not materially affect this estimate. No effect modification of HSV-2 by age, HPV-6/11 DNA, pregnancies, oral contraceptive use or cigarette smoking was observed. However, a significant interaction was detected between HSV-2 and HPV-16/18. Compared with women testing negative to both virus types, those positive for HSV-2 alone had a RR of 1.2 (95% CI = 0.9, 1.6), those positive for HPV-16/18 DNA alone had a RR of 4.3 (95% CI = 3.0, 6.0), and those positive for both viruses had a RR of 8.8 (95% CI = 5.9, 13.0). These findings corroborate recent laboratory evidence of a possible biological interaction between HSV-2 and HPV-16/18 in the development of cervical cancer. Further confirmatory studies are needed, given concerns with potential misclassification of exposure by the laboratory assays utilized.