In order to gain insight into the biology of the parasitic relationship between Heterodera schachtii and its host plant, it is important to understand the functional role of the nematode’s pharyngeal secretions. These secretions presumably play a key role in establishing and maintaining a feeding site for the nematode. An optimized method was used for the in vitro production of H. schachtii pharyngeal gland secretions. These pharyngeal secretions were mainly produced in an insoluble form and could be solubilized under denaturing conditions for further analysis. The soluble fraction was concentrated with StrataClean (Stratagene, La Jolla, CA) or with a phenol/ether extraction. These methods made it possible for the first time to separate the secreted proteins on two-dimensional gels. By combining a micropreparative sample preparation with mass spectrometry, two β-1,4-endoglucanases were identified. A third spot was identified as a novel protein by microsequencing. This is the first report on protein sequence information from pharyngeal secretions of a plant parasitic nematode.