Ants that had been completely isolated for three weeks were subjected to aggressive behavior, but not those that had continued to receive airflow from the mother colony. Adsorbing the nest volatiles from the airflow by SuperQ abolished this difference, with these ants now also being subjected to aggression, indicating that nest volatiles can modulate nestmate recognition. Reverse airflow also reduced the level of aggression but to a lesser extent than airflow directed from the mother colony. In queenless colonies the overall aggression was reduced under all regimes, and there was no effect of flow, suggesting that the volatiles involved are queen-borne. The SuperQ adsorbed volatiles originated from Dufour's gland secretions of both workers and queen, implicating them in the process. Cuticular hydrocarbon profiles were not affected by exposure to nest volatiles, suggesting that the latter either constitute part of the recognition cues or affect worker behavior via a different, as yet elusive mechanism.