Objectives: Suppressive herpes simplex virus (HSV) therapy can decrease plasma, cervical, and rectal HIV-1 levels in HIV-1/HSV-2 co-infected persons. We evaluated the effect of HSV-2 suppression on seminal HIV-1 levels. Design: Twenty antiretroviral therapy (ART)-naive HIV-1/HSV-2 men who have sex with men (MSM) in Lima, Peru, with CD4 >200cells/μl randomly received valacyclovir 500 mg twice daily or placebo for 8 weeks, then the alternative regimen for 8 weeks after a 2-week washout. Peripheral blood and semen specimens were collected weekly. Anogenital swab specimens for HSV DNA were self-collected daily and during clinic visits. Methods: HIV-1 RNA was quantified in seminal and blood plasma by TaqMan real-time polymerase chain reaction (RT-PCR) or Roche Amplicor Monitor assays. HSV and seminal cytomegalovirus (CMV) were quantified by RT-PCR. Linear mixed models examined differences within participants by treatment arm. Results: Median CD4cell count of participants was 424 cells/μl. HIV-1 was detected in 71% of 231 semen specimens. HSV was detected from 29 and 4.4% of swabs on placebo and valacyclovir, respectively (P< 0.001). Valacyclovir significantly reduced the proportion of days with detectable seminal HIV-1 (63% during valacyclovir vs. 78% during placebo; P=0.04). Seminal HIV-1 quantity was 0.25 log10 copies/ml lower [95% confidence interval (CI) -0.40 to -0.10; P = 0.001] during the valacyclovir arm compared with placebo, a 44% reduction. CD4 cell count (P=0.32) and seminal cellular CMV quantity (P=0.68) did not predict seminal plasma HIV-1 level. Conclusions: Suppressive valacyclovir reduced seminal HIV-1 levels in HIV-1/HSV-2 co-infected MSM not receiving ART. The significance of this finding will be evaluated in a trial with HIV-1 transmission as the outcome. © 2009 Wolters Kluwer Health|Lippincott Williams & Wilkins.