With the Nasonia vitripennis genome sequences available, we attempted to determine the proteins present in venom by two different approaches. First, we searched for the transcripts of venom proteins by a bioinformatic approach using amino acid sequences of known hymenopteran venom proteins. Second, we performed proteomic analyses of crude N. vitripennis venom removed from the venom reservoir, implementing both an off-line two-dimensional liquid chromatography matrix-assisted laser desorption/ ionization time-of-flight (2D-LC-MALDI-TOF) mass spectrometry (MS) and a two-dimensional liquid chromatography electrospray ionization Founer transform ion cyclotron resonance (2D-LC-ESI-FT-ICR) MS setup. This combination of bioinformatic and proteomic studies resulted in an extraordinary richness of identified venom constituents. Moreover, half of the 79 identified proteins were not yet associated with insect venoms: 16 proteins showed similarity only to known proteins from other tissues or secretions, and an additional 23 did not show similarity to any known protein. Serine proteases and their inhibitors were the most represented. Fifteen nonsecretory proteins were also identified by proteomic means and probably represent so-called ‘venom trace elements’. The present study contributes greatly to the understanding of the biological diversity of the venom of parasitoid wasps at the molecular level.