Sexual transmission accounts for the majority of new HIV infections worldwide with sexually exposed cervicovaginal and colorectal mucosae being primary sites of infection. Two recent Phase 1 rectal microbicide trials included, as an ancillary endpoint, suppression of ex vivo HIV infection of in vivo microbicide-exposed rectal mucosal tissue biopsies. Both trials demonstrated significant suppression of biopsy infectibility in drug-exposed versus placebo-exposed tissue. This potential early biomarker of efficacy has raised the feasibility of utilizing "snap-frozen" tissue samples, acquired at multiple trial sites to be shipped for central processing, providing a mechanism to correlate tissue drug concentrations with a functional index of HIV prevention. While previous reports have indicated acceptable comparability of fresh versus freeze-thawed cervicovaginal tissue samples, no similar evaluations with colorectal tissue biopsies have been done. In this study, rectal biopsies from healthy, HIV-seronegative participants were assessed for structural integrity (histology), viability (MTT assays), and tissue infectibility to compare results from fresh versus combinations of freeze/thaw protocols. Results indicated that while all protocols showed equivalent viability with fresh samples (MTT), histology documented poor preservation of tissue integrity following freezing. Infectibility results from freeze-thawed colorectal tissue were markedly lower (usually<25% of fresh samples) and varied greatly and unpredictably. Centralized colorectal tissue infectibility assays using biopsies from remote trial sites cannot currently be supported under these protocols.