Profilin, an actin-binding protein, was previously described as a ubiquitous allergen which is responsible for cross-reactivities in about 20% of pollen and food allergic patients. A complete cDNA clone coding for timothy grass (Phelum pratense) pollen profilin was isolated using allergic patients IgE. The deduced amino acid sequence of timothy grass profilin shares a sequence identity of 79% with birch profilin and other plant profilins and a lower average sequence identity of 35% with other eukaryotic profilins. The high degree of homology among different plant profilins at the DNA and protein level explains the extensive cross-reactivities observed in profilin allergic patients. Recombinant timothy grass pollen profilin was expressed in Escherichia coli as a beta-galactosidase fusion protein and shown to bind IgE from profilin allergic patients similar to recombinant birch profilin. Slight differences regarding the IgE-binding capacity of birch and timothy grass profilin indicate that not all IgE-epitopes of the two profilins are conserved. It is speculated that profilin allergic patients were initially sensitized against a certain profilin and then cross-react with the homologous proteins.