Rye grass (Lolium perenne) pollen extracts were fractionated by gel filtration (Sephadex G-25, G-50, and G-75) and ion-exchange chromatography (DEAE-Sephadex). The allergenic activity of the pollen fractions was assessed by both in vivo (monkey passive cutaneous anaphylaxis; PCA) and in vitro (inhibition of the radioallergosorbant test; RAST) techniques. A rye grass pollen allergen was isolated in both monomeric (MW 33,000) and dimeric (MW 64,000) forms. While the latter preparation exerted the greatest allergenic activity in vivo, the activity of both components was equal when tested by RAST inhibition. On the other hand, a low molecular weight allergen was detected in the pollen extract which, although it exerted significant activity in the RAST inhibition test, was only weakly active in monkey PCA. It was concluded that the antigenic valence of the allergen exerted a strong influence on its in vivo activity, but that this factor was of little importance in the in vitro test.