In dequeened honeybee colonies ovarian activation occurs in some workers, and the pheromonal bouquets of these laying workers become more queen-like. In the Asiatic honeybee, Apis cerana, we compared the amount of 9-keto-2(E)-decenoic acid (9-ODA), a mandibular gland pheromone component, between non-laying workers from queenright colonies and laying workers from queenless colonies, and further, applied synthetic 9-ODA to workers to determine whether they discriminate workers with activated ovaries based on the level of this compound. Levels of 9-ODA were higher in laying workers from dequeened colonies than in non-laying workers from queenright colonies. In both queenright and queenless colonies, workers attacked more workers treated with 9-ODA than control-treated workers. These results suggest that detection of pseudoqueens in A. cerana is mediated by changes in 9-ODA.