Honey bees undergo a physiological transition from nursing to foraging approximately 21 days after adult emergence. This transition is delayed by ethyl oleate (EO), a primer pheromone produced by foragers when exposed to ethanol from fermented nectar. We demonstrate here that two secreted α/β-hydrolases (BeeBase ID: GB11403 and GB13365) are responsible for the reversible esterification of ethanol with oleic acid, giving EO. Expression of hydrolase GB11403 was shown to be significantly up-regulated in foragers, relative to nurses. Tissue perfusion experiments with labeled substrates consistently localized the highest level of EO production in the head, whereas in situ imaging revealed expression of relevant EO biosynthetic genes and enzymatic activity along the esophagus, the site of ethanol exposure during nectar intake. Both α/β-hydrolases were expressed in Pichia pastoris, purified and were shown produce EO in vitro. Experiments with live bees fed ethanol demonstrated that EO formed in regurgitate accumulates in the honey crop and exudes to the exoskeleton, from where it exerts its primer effect on younger bees.