Mucosal tissue is the main portal of entry for HIV-1 infection and, in macaques, has been demonstrated to be a significant compartment for viral replication and CD4+ T lymphocyte depletion. Quantitating tissue viral burden in addition to plasma viral load provides insights into HIV-1 pathogenesis and an additional means to gauge antiretroviral response. The aim of this study was to develop reliable, reproducible, and sensitive assays to quantitate tissue viral burden of HIV-1 RNA and DNA using 1-3 endoscopically acquired, rectosigmoid biopsies. Total DNA and RNA were simultaneously extracted following homogenization from the same tissue samples. Quantitative polymerase chain reaction (PCR) assay in the HIV-1 LTR region was used to detect viral DNA and RT-PCR for viral RNA. It was determined that HIV-1 RNA and DNA can be reproducibly quantified from a single rectosigmoid biopsy with minimal intra-assay or intra-patient variability. These results reflect high recovery of extracted nucleic acids with calculated results accurately reflecting in vivo levels. The techniques outlined differ from currently available approaches by incorporating control standards to identify loss or degradation of RNA and DNA from acquisition through the in vitro assay and permit extraction with high yields of RNA and DNA from the same tissue sample.