An allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated by a combination of copper chelate affinity chromatography and ion exchange chromatography. Phl p V binds IgE from serum of grass-sensitized donors as revealed in immunoelectrophoretic techniques and in SDS-PAGE immunoblot, and luminescence immunoassay (LIA) inhibition experiments indicate that the allergen represents a significant part of the IgE binding capacity of the extract. In immunoelectrophoresis, Phl p V is revealed as a single precipitate. However, molecular weight studies show that Phl p V consists of at least two isoforms with similar immunochemical properties, but with different molecular size. After SDS-PAGE treatment purified Phl p V is identified as two IgE-binding components, Phl p Va and Phl p Vb, with molecular weights 33 and 29 kD. After HPLC gel filtration, Phl p Va and Phl p Vb are identified in the major 30-kD eluate. After Sephadex G75 gel filtration of whole pollen extract, Phl p V is identified in fractions corresponding to molecular weights 47 and 25 kD. The 47-kD fraction corresponds to Phl p Va/Phl p Vb as seen in SDS-PAGE, while the 25-kD component presumably corresponds to a degradation product present in whole pollen extract. The NH2-terminal sequence of Phl p V, corresponding to approximately 10% of the molecule, has been determined. The sequence shows minor variations in some residues and contains besides many alanine residues also hydroxyproline; the sequence reveals no homologies to any known NH2 terminal sequence of other proteins. The amino acid composition, revealing 26 mole % alanine and no cysteine, does not show any similarities to other known amino acid compositions of allergens. From the amino acid composition determination and an immunoelectrophoretic comparison, Phl p V is estimated to represent 6% (w/w) of the whole pollen extract.