Lymphocyte populations, adhesion molecule and cytokine expression were determined in lymph nodes draining peripheral (popliteal and prescapular) or mucosal (abomasal and jejunal) tissue sites using flow cytometry analysis, immunostaining and cytokine-specific reverse-transcription-polymerase chain reaction (RT-PCR). Similar proportions of lymphocyte subpopulations were present in all lymph nodes except for immunoglobulin A+ (IgA+) B cells which were only present in significant numbers in the gastrointestinal lymph nodes. Peripheral lymph nodes contained a significantly higher number of CD4+ cells expressing L-selectin and beta 1-integrin than mucosal lymph nodes while the alpha 4-integrin chain was expressed at similar levels in all lymph nodes. The peripheral node adressin recognized by the MECA 79 monoclonal antibody (mAb) was mainly expressed on peripheral lymph node vessels. RT-PCR analysis showed that interleukin (IL)-10 and IL-4 were preferentially induced in the gastrointestinal lymph nodes while IL-2 and interferon-gamma (IFN-gamma) were induced in all lymph nodes after polyclonal stimulation. These results indicate that there are substantial differences in the cell populations and microenvironments of lymph nodes draining mucosal and peripheral tissue sites in adult sheep.