Earlier studies have shown that a substance(s) released from the egg jelly of the toad Bufo arenarum is required for fertilization. In this paper some properties of this diffusible factor were further examined, and a procedure was designed for its isolation from crude egg extracts. The active component is soluble in water and ethanol, and insoluble in chloroform, ether and n-butanol. The biological activity is stable to liophylization and to heat, and remains unaffected after trypsin treatment. In contrast, it is impaired after treatment with ethyl acetate, 0.1 N HCl or chloroform, and is completely destroyed after converting the diffusible factor into ash. Data are presented showing that the recovery of fertilizability of extracted eggs in the bioassay system as carried out under present conditions, cannot be ascribed to a pH alteration of the insemination medium. This lends further support to the view that diffusible factor activity is not mediated through a pH effect. The factor was purified by gel chromatography coupled with desalting and paper chromatography. The active molecule is of low molecular weight and appears associated with a high pH ninhydrin-positive fraction.