Stein et al. (1991) identified a soluble, extracellular factor that induces ventral structures at the site where it is injected in the extracellular space of the early Drosophila embryo. This factor, called polarizing activity, has the properties predicted for a ligand for the transmembrane receptor encoded by the Toll gene. Using a bioassay to follow activity, we purified a 24 × 10(3) M(r) protein that has polarizing activity. The purified protein is recognized by antibodies to the C-terminal half of the Spatzle protein, indicating that this polarizing activity is a product of the spatzle gene. The purified protein is smaller than the primary translation product of spatzle, suggesting that proteolytic processing of Spatzle on the ventral side of the embryo is required to generate the localized, active form of the protein.