Mycobacterium tuberculosis infection continues to be a daunting clinical challenge. Although it may well be one of the most studied bacteria in history, several aspects of its pathology remain a mystery. The resurgence of drug-resistant M. tuberculosis strains and with its unusual pathology have promoted a renewed basic and clinical research interest in developing new therapies to combat this pathogen. The primary localization site for M. tuberculosis is within alveolar macrophages. Drug delivery strategies and novel therapeutic agents designed to target alveolar macrophages may lead to efficient destruction of M. tuberculosis. Oligodeoxynucleotides (ODN) are short segments of nucleic acids that can interfere with transcription and translation processes. In this report, a monocyte-macrophage cell line was characterized in regard to ODN transport in the presence or absence of M. tuberculosis infection. The cells accumulated ODN in a time-dependent and concentration-dependent manner, regardless of the presence of serum. After 4 hours of incubation with M. tuberculosis (multiplicity of infection [MOI] 10:1), infected NR8383 cells demonstrated 1.5-7-fold increase in fluorescein isothiocyanate (FITC)-labeled phosphorothioate ODN accumulation as measured by flow cytometry. The increase in uptake was associated only with fluorescent-labeled ODN and not labeled markers of fluid phase endocytosls (e.g., tetramethylrhodamine isothiocyanate [TRITC], FITC-labeled dextran). NR8383 cells activated by phytohemagglutinin (PHA) did not demonstrate a significant increase in the uptake of either FITC-labeled dextran or FITC-labeled ODN. These studies demonstrate that NR8383 cells that have been infected with M. tuberculosis can specifically accumulate ODN, and this route of accumulation may lead to a means of drug targeting to mycobacteria-containing cells.