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We have identified saponins in the root of Polygala senega L., a plant indigenous to the Canadian prairies, which display immunopotentiation activity to protein and viral antigens. By two-step extraction and hemolytic activity-guided fractionation by silica flush chromatography six saponin fractions were generated and their HPLC profiles determined. Two dominant fractions, designated as PS-1 and PS-2, were tested for adjuvant activity in mice immunized with ovalbumin, and hens immunized with rotavirus. The resulting adjuvant activity was compared with that of Quil A saponin. The P. senega saponins increased specific antibody levels to the antigens, in both mice and hens. In mice, there was a preferential increase of the IgG2a subclass, and upon in vitro secondary antigen stimulation, high IL-2 and IFN-gamma levels were observed in spleen cell cultures from P. senega saponins-immunized animals. The saponins were tested for their toxicity by lethality in mice and were found to be less toxic at the same dose than their counterpart Quil A. The results of this study indicated the potential of P. senega saponins as vaccine adjuvants to increase specific immune responses.