AbstractLeafcutter ants live in symbiosis with fungi they grow for food and different bacteria they use for protection against fungal parasites. Some workers are covered with a layer of bacteria like Pseudonocardia, but the behavior of these workers has been the subject of little research. In order to obtain a clearer understanding of this phenomenon in Acromyrmex octospinosus, two experiments were conducted with majors, minors, workers with little bacterial layer and workers with a well-developed bacterial layer. In a first step, scan sampling was used to distinguish the preferred location of workers with bacterial coats, compared to the preferences of their nestmates. Finally, 21 behavior patterns of workers with a bacterial layer on their integument were investigated through focal observations and compared to the behavior of their nestmates. Our findings suggest that workers covered with a high amount of Pseudonocardia differ from their sisters in their preference of location, in that they significantly prefer the bottom part of the nest and cannot be found outside the fungus chamber in the feeding area or the waste area. Additionally, these ants show more fungus grooming and are groomed by nestmates more often than the other workers. These results suggest that workers with an external bacterial layer evolved behavioral specialization.