2-Alkanones (C7 and C9) and 3-hydroxyalkanoic acids (C8 and C10) were identified in worker mandibular glands of three Japanese Bombus species. In Apis species, 2-heptanone is regarded as one of alarm pheromones. In Apis cerana foragers, 3-hydroxyotanoic acid was identified as a major mandibular acid with a small amount of 2-heptanone, whereas foragers of Apis mellifera produce nearly 1 microgram of 2-heptanone with a trace of 3-hydroxyoctanoic acid in their mandibular glands. Application of deuterated 3-hydroxyalkanoic acids onto mandibular glands resulted in detection of deuterated 2-alkanones. Thus, 2-alkanones are biosynthesized through oxidative decarboxylation from corresponding 3-hydroxyalkanoic acids in the mandibular glands. Possible precursors for 3-hydroxyalkanoic acids were examined in the same way.