Using two- and three-color flow cytometry, we investigated the surface makers of regional lymph node lymphocytes (RLNL) in 54 patients with primary lung cancer in order to determine whether or not RLNL are in an activation state when compared with the corresponding peripheral blood lymphocytes (PBL) for the purpose of clarifying the characteristics of RLNL. RLNL showed a cell composition that was different from the corresponding PBL with a high proportion of CD3+ cells, CD4+ cells and CD20+ (B) cells as well as a low proportion of CD8+ cells and CD16+ (NK) cells. RLNL also contained a significantly higher proportion of CD45RO+ T cells and a lower proportion of CD45RA+ T cells in comparison to the corresponding PBL. Furthermore, we explored the activation-related molecules such as the interleukin-2 receptor (IL-2R) alpha chain, the IL-2R beta chain, HLA-DR and leukocyte function-associated antigen 1 (LFA-1) on CD4+ and CD8+ cells. The data showed that the expression of CD45RO, the IL-2R alpha chain, HLA-DR on CD4+ cells, and those of CD45RO and HLA-DR on CD8+ cells were significantly higher in RLNL than in PBL. On the other hand, PBL showed a higher expression of the IL-2R beta chain and LFA-1 only on CD8+ cells, which are thought to include CD8+ NK cells. When these activation related molecules were analyzed on CD45RO+ T cells, which are thought to be memory T cells, then the expression of the IL-2R alpha chain, HLA-DR on CD4+CD45RO+ cells and HLA-DR on CD8+CD45RO+ cells were significantly higher in RLNL than in PBL. Lastly, we analyzed the surface molecules according to such clinical factors as metastasis to the lymph nodes and the stage of lung cancer. Interestingly, RLNL with lymph node metastasis showed an increase in the percentage of CD20+ cells and a decrease in the percentage of CD4+ cells in comparison to those with benign lung disease, while these differences were not observed in PBL. The expression of ++CD45RO and LFA-1 on CD4+ and LFA-1 on CD8+ cells on RLNL in stage I + II is significantly higher in comparison with benign lung disease. Furthermore, RLNL in stage III + IV revealed the reduced expression of CD45RO, HLA-DR and LFA-1 on both CD4+ and CD8+ cells compared with those in stage I + II. These results thus demonstrated that RLNL were in a more activated state, particularly in stages I + II, than PBL. Whether these activation states in RLNL are directed against autologous tumor cells or not can hopefully be elucidated after the completion of further ongoing studies.