Although there is discordance between human immunodeficiency virus (HIV) blood plasma and seminal plasma viral loads (VL), little is known about the dynamics of VL rebound in these compartments upon discontinuation of highly active antiretroviral therapy (HAART). Therefore, we sought to examine the relationship between blood and semen VL rebound after discontinuation of HAART. Participants in this substudy were men enrolled from two centers of a multicenter, placebo-controlled randomized trial of HIV therapeutic vaccination using ALVAC with or without Remune. With at least 2 years of sustained virologic suppression and following a 20-week vaccination course, subjects underwent structured HAART interruption. Fourteen men provided semen samples. Seven to 12 weeks after HAART interruption, all 14 men had detectable blood VLs whereas 8 of 14 had detectable seminal VLs. There was a significant correlation between blood and seminal VLs (Spearman r=0.58, p=0.03) at the time of semen collection. An earlier time to detectable blood VL after HAART interruption was associated with higher seminal VL (Spearman r=-0.64, p=0.02). These findings support the compartmentalization of HIV and underscore the importance of understanding the genital tract as an HIV reservoir in the quest to minimize HIV transmission.