Synergistic activity (SA), which is the capacity to support the in vitro proliferation of High Proliferation Potential Colony Forming Cells (HPP-CFC's), has been fractionated from conditioned media by standard biochemical techniques. On fractionation of both Wehi conditioned medium (Wehi CM) or human placental conditioned medium (HPCM) by several techniques, multiple peaks of SA were observed. Protein from HPCM expressing SA, ranged from 9,000-28,000 in molecular weight as determined by gel filtration. Chromatofocusing crude HPCM separated two activities possessing isoelectric points of 5.7 and 5.3. In crude Wehi CM, inhibitors or toxicity occasionally masked SA which partially adsorbed to Con-A sepharose and could be eluted with competing sugar. Further fractionation of this material on DEAE-sepharose separated three peaks of activity. Pooling the active fractions from the first two peaks gave a preparation that was up to 179 fold more active than the crude material.