During the serial passage of Mycobacterium bovis bacillus Calmette–Guérin (BCG) in different countries after initial seed distribution from the Pasteur Institute, specific insertions and deletions in the genome among BCG substrains were observed and speculated to result in differences in immunological activities. ‘Early-shared strains’ of BCG (Russia, Moreau, Japan, Sweden, Birkhaug), distributed by the Pasteur Institute, which conserve three types of mycolate (α, methoxy, keto) in cell wall, exhibited stronger activities of induction of nitric oxide, interleukin-1β (IL-1β), IL-6, IL-8, IL-12 and tumor necrosis factor (TNF)-α, from human epithelial cell line A549, human myelomonocytic cell line THP-1 and mouse bone marrow cells in the presence of interferon-γ (IFN-γ) than did ‘late-shared strains’ of BCG (Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia, Pasteur). The stronger induction of IL-12 and TNF-α in the presence of IFN-γ was also observed by trehalose 6,6′-dimycolate (TDM) extracted from BCG-Japan than by TDM from BCG-Connaught, which lacks the methoxymycolate residue. These results suggest that ‘early-shared strains’ are more potent immunostimulating agents than ‘late-shared strains’, which could be attributed partially to methoxymycolate. Our study provides the basic information for immunological characterization of various BCG strains and may contribute to a re-evaluation of them as a reference strain for vaccination against tuberculosis.