It is of critical importance to precisely understand the modalities of HIV presence in semen, especially with regard to procreation. In this study, paired blood and semen samples from 31 human immunodeficiency virus type 1 (HIV-1)-positive men were assessed for cell-free HIV-RNA load in blood plasma (BP) and seminal plasma (SP), and for detection of HIV by culture, PCR and RT-PCR in semen cellular fractions separated by centrifugation on Percoll gradient. HIV-RNA was detected in 94% of BP and 84% of SP samples. For 11 men (35%), HIV-RNA load in SP was equal or superior to that observed in blood. HIV-DNA presence was demonstrated (either by PCR or culture positivity) in 39% of the non-spermatozoal cells (NSC)-enriched fractions, and in one Percoll-selected sperm pellet. HIV-RNA was detected in 17% (4/23) of the sperm pellets. This positivity was associated with an HIV-RNA load in SP equal or superior to the HIV-RNA load in blood, a high rate of HIV-DNA detection in the NSC fraction, and a low CD4+ cell count. In such conditions, a significant viral production inside the genital tract is more likely to be present, and a close association between HIV and gametes might occur. Assisted procreation with selected spermatozoa should be preceded by accurate assessments of viral presence in blood, SP and semen cellular fractions.