To determine whether human whole semen (WS) and seminal plasma (SP) either previously frozen or freshly acquired altered ex vivo infectibility of human colonic explants or was associated with histology or toxicity changes, which may influence mucosal HIV-1 transmission in vivo. Pooled human semen samples were freshly obtained from study volunteers (never frozen) and from commercial sources (frozen/thawed). Endoscopically acquired rectal biopsies were evaluated for toxicity following titered ex vivo WS/SP exposure by histological grading and by MTT assay. The ex vivo HIV-1 biopsy challenge model was used to evaluate effects of exposure to either previously frozen or freshly acquired WS/SP on HIV infectibility at a range of viral inocula (10-10 TCID). To evaluate the effects at lower viral inocula of HIV-1 (10-10), experiments in the presence or absence of WS/SP were also performed utilizing TZM-bl cells. MTT assays and histological scoring demonstrated no tissue degradation of biopsies when exposed for 2 h to concentrations of 10% or 100% of either fresh or previously frozen WS/SP. Ex vivo biopsy HIV-1 challenge experiments showed no differences in the presence of freshly acquired or previously frozen/thawed WS/SP compared with control; no differences were seen with lower infectious titers on TZM-bl cells. Within the limits of assay sensitivity and variability, these data show no toxicity or significant enhancement of HIV-1 infectibility of human rectal mucosa using the colorectal explant model with either pooled fresh or frozen/thawed nonautologous human semen.