Two molecular species of lipoteichoic acid (LTA 1 and LTA 2) were isolated from whole cells of Streptococcus faecalis (Enterococcus hirae) ATCC 9790 by hydrophobic chromatography on Octyl Sepharose CL-4B. Chemical analysis revealed that LTA 1 and LTA 2 contained two and four acyl lipid anchors respectively. LTA 1 was less active than LTA 2 in inducing cytokines, except interleukin-1 (IL-1), but their in vivo antitumour effects were similar. LTA 2 was a potent inducer of tumour necrosis factor (TNF) and interferon (IFN) production and had excellent antitumour activity against Meth A fibrosarcoma established in mice. Deacylation of LTA 2 by alkaline hydrolysis abolished these biological activities. The phosphatidylglycolipid fraction derived from LTA 2 after acid hydrolysis could also induce TNF, IFN, and IL-1 production, as well as having antitumour activity against Meth A fibrosarcoma. Therefore, the lipid anchor portion of S. faecalis LTA may play an important role in the manifestation of these various biological activities.