Ticks (Ixodoidea) likely transmit the greatest variety of human and animal pathogens of any arthropod vector. Despite their medical significance little data is available about the messenger molecules in the central nervous system that coordinate all physiological processes in these animals, including behaviour. In our study, we performed the first comprehensive neuropeptidomic analysis of a tick species by using MALDI-TOF mass spectrometry. Specifically we analyzed the neuropeptides in the synganglion of Ixodes scapularis. The forthcoming sequence of the genome of this species will represent the first genomic analysis of a member of the large subphylum Chelicerata. For our approach we used information from predicted neuropeptide precursor sequences found in EST databases [Christie, AE. Neuropeptide discovery in Ixodoidea: an in silico investigation using publicly accessible expressed sequence tags. Gen Comp Endocrinol 2008;157:174-185] as well as data obtained by complete de novo sequencing. The direct tissue profiling yielded 20 neuropeptides from 12 neuropeptide precursors. The sequences of these neuropeptides are not as unique as predicted; a comparison with the peptidome of other invertebrates shows a close relationship with insect neuropeptides. This work will provide a resource for studying tick neurobiology and will hopefully also help to identify novel targets for tick and tick-borne disease control.