Honey bee (Apis mellifera L.) queens mate early in life and store sperm for years. Male bees likely contribute significantly to sperm survival. Proteins were extracted from seminal vesicles and semen of mature drones, separated by electrophoresis, and analysed by peptide mass fingerprinting. Computer searches against three databases, general species, honey bees and fruit flies, were performed. Spectra were used to query the recently generated honey bee genome protein list as well as general species and fruit fly databases. Of the 69 unique honey bee proteins found, 66 are also in Drosophila melanogaster. Two proteins only matched honey bee genes and one is a widespread protein lost from the fly genome. There is over-representation of genes implicated in the glycolysis pathway. Metabolism-associated proteins were found primarily in the seminal vesicle. Male accessory gland proteins as identified in Drosophila rarely had orthologs among proteins found in the honey bee. A complete listing of gel spots chosen including honey bee genome matches and Mascot searches of MALDI-TOF results with statistics is in the Supplementary table. MALDI-TOF spectra and more complete Mascot peptide mass fingerprinting data are available on request. Supplementary figs 1-3 show the stained protein gels.