Adjuvant activity of saponins extracted from the South American tree Quillaja saponaria has been demonstrated with many antigens. Recently, four saponin fractions (designated as QS-7, QS-17, QS-18, and QS-21) with adjuvant activity were purified by reverse phase chromatography. In particular, efficacy of the less toxic QS-21 fraction has been demonstrated with several recombinant viral antigens including HIV gp120. Here, we report a novel saponin fraction (designated as QS-L1) derived from Quillaja saponaria. Unlike previously identified saponins, QS-L1 had a different chemical structure and showed adjuvant activity only when administered in the presence of alum-precipitated antigen. Interestingly, the QS-L1 greatly increased not only a humoral immune response but also cellular immune response to recombinant hepatitis B virus surface antigen (HBsAg). Furthermore, QS-L1 showed lower toxicity in vivo and in vitro than the previously identified saponin fraction, QS-21. Finally, we examined the chemical structure of the QS-L1 using mass spectroscopic analysis, carbohydrate composition analysis and NMR spectroscopic analysis. Thus, our results indicated that this novel QS-L1 saponin fraction had several desirable properties required for an effective adjuvant.