Our initial study of honeybees using the rapid Golgi method showed that dendritic spines on calycal interneurons had shorter stems due to spine head enlargement in bees with greater cumulative experience. This study sought to determine if spine stem shortening could be induced rapidly during the first orientation flight, a one-trial place learning event. Newly emerged bees were reared in a small broodless hive with a virgin queen and allowed to take their first orientation flight at 6 and 8 days of age. Spine profiles of 5 flyers and 5 non-flyers were traced in large scale using light microscopy and a modified camera lucida. Overall spine length and stem length were measured on these tracings using a digitizing tablet. Additional measurements of maximum spine head width, profile area, and perimeter were made using computer image analyses. Examining group differences in spine stem length as a function of overall spine length, our results revealed a clear association between rapid spine stem shortening and the first orientation flight lasting several minutes. This effect, however, was restricted to only the long spines. Flight-induced stem shortening was accompanied by elongated swelling of the spine head without an appreciable expansion of the spine perimeter.