Sperm are often stored, for a long time after mating, in females of various animal species. In case of the queen honeybee (Apis mellifera), sperm remain fertile for several years in the spermatheca. Little information is available regarding the effect of long-term storage of sperm on its fertility. To evaluate this, enzymes and/or sperm have been analysed from the spermatheca of 75 queens of various ages (0 year Y0, n=14; one year Y1, n=14; two years Y2, n=7; virgin queen VQ, n=40) and semen samples have been taken from 46 drones. The sperm from the spermatheca of older queens move more slowly (F=11.45, P < 0.0001) and show different movement patterns (Chi2=90.0, P < 0.0001) from those of the other groups. The spermatheca content of differently aged mated queens differ significantly with respect to the activities of lactate dehydrogenase (F=3.37, P < 0.05), citrate synthase (F=6.24, P < 0.005) and arginine kinase (F=9.44, P < 0.0006). Glyceraldehyde 3-phosphate dehydrogenase (F=0.10, P=0.91) does not differ significantly. The results suggest considerable changes in the energy metabolic profile of the spermatheca tissue, of the sperm or of both during sperm storage.