HIV-1 prevalence is approximately 23% among men working at a sugar estate in Malawi. Given the scale of the HIV epidemic in this country, it is important to determine possible cofactors of infection. The authors investigated associations between HIV-1 prevalence and herpes simplex virus 2 (HSV-2), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections (indicated by anti-HSV-2, anti-HCV and HBsAg positivity, respectively) in a nested case-control study of 279 HIV-positive and 280 HIV-negative male workers. The prevalence of HSV-2 infection was 88.1% among HIV-positive persons and 64.3% among HIV-negative control subjects (p <.01). This difference persisted after adjusting for sexual behavior and history of sexually transmitted diseases (OR = 4.12; 95% CI, 2.21-7.68). The prevalence of HCV seropositivity was 12.7% among HIV-positive persons and 10.0% among control subjects (p =.31), whereas that of HBV infection was 16.9% among HIV-positive persons and 14.4% among control subjects (p =.46). HSV-2 infection is significantly associated with prevalent HIV-1 infection in this population. Therefore, preventive measures for HSV-2 and HIV infection should be emphasized.