Penetration of antiretroviral drugs into the genital tract is crucial for virological efficacy in this compartment and impacts the transmission of the virus. Anti-HIV drugs showed different degrees of passage into the male and female genital systems, and their efficacy appeared related to the concentrations reached at the site of action. Raltegravir (RGV) is a novel drug whose target is the HIV preintegrase complex. Jones and colleagues demonstrated a significant passage in the cervicovaginal fluid of female healthy volunteers, with concentrations around 93% of plasma and a longer elimination half-life (17 versus 7 h). A paper recently published in this journal showed that males treated with RGV-containing regimens had undetectable viral loads in seminal plasma and showed variable but high drug penetration in such compartment, but no data of RGV penetration in seminal plasma over the whole dosing interval are currently available. We performed a multiple-dose, one-way, open-labeled, noncontrolled, single-center study. Eight healthy male volunteers were enrolled after written informed consent was obtained. Subjects were administered RGV (400 mg twice daily) in fed state for 4 days plus a single dose on day 5. On day 5, peripheral blood and semen (after 5 days of sexual abstinence) were concomitantly collected within 11 to 12 h (4 subjects) or 2 to 4 h (4 subjects) after last RGV dose intake. RGV blood plasma and seminal plasma levels were measured by a validated high-performance liquid chromatography (HPLC-PDA) method with limit of detection of 11.7 ng/ml. In the group with measurements performed at the end of the dosing interval, blood and semen RGV concentrations were 151 (51 to 443) and 937 (212 to 3,582) ng/ml, respectively, with a semen-to-blood plasma concentration ratio of 6.45 (3.78 to 9.37). In the group where RGV was measured after recent drug intake (2 to 4 h), plasma and semen concentrations were 651 (438 to 1,020) and 953 (541 to 1,706) ng/ml, respectively, with a semen-to-blood plasma ratio of 1.62 (1.33 to 2.06). All samples were found to be above the 95% inhibitory concentration of wild-type HIV-1 strains (14.6 ng/ml). Our study confirmed a high and variable degree of RGV penetration in seminal plasma. However, we showed for the first time that the magnitude of penetration significantly varies over the dosing interval. The semen-to-blood plasma concentration ratio was higher at the end of the dosing interval (6.45) than at 2 to 4 h after drug intake (1.62). This finding suggests a possible delay of drug penetration from plasma to semen and/or a slower RGV clearance in the compartment, leading to increasing levels in semen over time compared to stable plasma concentrations. The possible therapeutical and prophylactic implications warrant study in the clinical setting.