The honeybee is the economically most important beneficial insect and a model for studying immunity, development and social behavior. Hence, this species was selected for genome sequencing and annotation. An intensive interplay between bioinformatics and mass spectrometry (MS) resulted in the annotation of 36 neuropeptide genes (Hummon et al., 2006). Exactly 100 peptides were demonstrated by a variety of MS techniques. In this follow-up study we dissected and analysed separately all ganglia of the central nervous system (CNS) of adult worker bees in three repeats. The combined MALDI-TOF spectra enabled the accurate mapping of 67 peptides, encoded by 20 precursors. We also demonstrated the expression of an additional but already predicted peptide. In addition to putative bioactive peptides we also list and discuss spacer peptides, propeptides and truncated peptides. The majority of such peptides have a more restricted distribution pattern. Their presence provides some information on the precursor turnover and/or the location of neural cell bodies in which they are produced. Of a given precursor, the (neuro)-peptides with the widest distribution pattern are likely to be the best candidates to interact with receptors. The separate analysis of a neuroendocrine complex and the mushroom body yields suggestions as to which (neuro)-peptides might act as hormones and which neuropeptides might be involved in the complex spectrum of non-hormone driven honeybee behaviour, at these sites. Our data complement immunohistochemical studies of (neuro)-peptides in the honeybee, and form a reference for comparative studies in other insect or arthropod models, in particular in the light of recent or upcoming genome projects. Finally, they also form a firm basis for physiological, functional and/or differential peptidomics studies in the honeybee.