Immunological properties of synthetic 6-O-mycoloyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (6-O-mycoloyl-N-acetylmuramyldipeptide) and 6-O-mycoloyl-N-acetylmuramic acid were examined in guinea pigs and mice in comparison with those of BCG cell-wall skeleton, N-acetylmuramyl-L-alanyl-D-isoglutamine, and 6-O-stearoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine. 6-O-Mycoloyl-N-acetylmuramyldipeptide showed a potent adjuvant activity for the induction of delayed type hypersensitivy to N-acetyl-L-tyrosine-3-azobenzene-4'-arsonic acid (ABA-N-acetyltyrosine). It was also found that 6-O-mycoloyl-N-acetylmuramyldipeptide treated with oil droplets or suspended in phosphate-buffered saline was as effective as oil-attached BCG cell-wall skeleton for the generation of cell-mediated cytotoxic effector cells to mastocytoma P815-X2 cells in the spleen of C57BL/6J mice in vivo. However, 6-O-mycoloyl-N-acetyl-muramyldipeptide was less active as adjuvant than BCG cell-wall skeleton and N-acetylmuramyldipeptide in enhancing the circulating antibody formation to T-independent antigen, 2,4-dinitrophenyl-lysyl (DNP-Lys)-Ficoll in vivo, and on the generation of helper function of carrier-primed T-cells, and was inactive as a mitogen on normal mouse spleen cells. On the other hand, although 6-O-mycoloyl-N-acetylmuramic acid was shown to be inactive as adjuvant on immune systems described above, it was active as a mitogen on normal mouse spleen cells.