It is becoming increasingly obvious that evaluation of a vaccine aimed at preventing HIV infection should include assessment of induced immunity at mucosal sites of viral entry. Among the most salient immune responses are viral-specific antibodies. A recent report on IgA-secreting plasma cells in human duodenal explants prompted us to examine similar duodenal and rectal biopsies of rhesus macaques, a key animal model for pre-clinical HIV/SIV vaccine studies, and characterize the local resident B-cells. Here we report that non-human primate rectal explants possess similar levels of B-cells as duodenal explants. We characterize the antibody isotype expression on mucosal memory B-cells and show for the first time that the B-cell memory subsets of the duodenum and rectum are distinct from those of PBMC, not only by essentially lacking CD27⁺ cells, as previously reported for uninfected macaques (Titanji et al., 2010), but also in being mostly IgD⁻. SIV- and SHIV-infected macaques had fewer total IgA-secreting cells in rectal tissue compared to naïve macaques. As expected, the fractions of B-cells with surface expression of IgA were dominant in the rectal and duodenal explants whereas in PBMC IgG surface expression was dominant among IgD⁻ B-cells. Mucosal antibody secreting cells were found to be predominantly plasma cells/plasma blasts based on their lack of response to stimulation. Importantly, short-term culture of rectal explants of SIV- and SHIV-positive animals led to secretion of Env-specific IgA into the culture supernatant which could be easily measured by ELISA. Collection of such culture supernatant over several days allows for accumulation of mucosal antibody in amounts that should enable antibody purification, characterization, and use in functional assays. Rectal explants can be readily obtained and unequivocally identify the mucosal tissue as the source of antibody. Overall they facilitate evaluation of mucosal vaccines.