Arabino-mycolates are components of the cell-wall skeleton of Mycobacterium bovis BCG (BCG-CWS). It is known that synthesized arabinomycolates induce the production of tumor necrosis factor alpha (TNF-α) in murine macrophage cell lines at an intensity similar to that of BCG-CWS. However the immunological activity of natural arabino-mycolates isolated from BCG has not been investigated, probably due to the complexity of the molecule. In this paper, we investigated the immunostimulatory activity of arabino-mycolates isolated from BCG-CWS by acid hydrolysis. Arabino-mycolates obtained by acid hydrolysis from the originally prepared CWS (SMP-105) of M. bovis BCG Tokyo 172 strain consisted mainly of mono-arabinose mono-mycolate, penta-arabinose tetra-mycolate and hexa-arabinose tetramycolate fractions. Arabino-mycolates significantly induced TNF-α production with an intensity comparable to that of CWS and enhanced delayed type hypersensitivity (DTH) reactions against inactivated tumor cells. Arabino-mycolates-induced TNF-α production was completely dependent on TLR2 and MyD88 pathways. These findings indicate that isolated natural arabino-mycolates possess potent adjuvant immunostimulatory activity.