Nestmate recognition is a critical element in social insect organization, providing a means to maintain territoriality and close the colony to parasites and predators. Ants detect the colony chemical label via their antennae and respond to the label mismatch of an intruder with aggressive behavior. In the fire ant, Solenopsis invicta, worker ability to recognize conspecific nonnestmates decreases if the colony queen is removed, such that they do not recognize conspecific nonnestmates as different. Here, we tested the hypothesis that the presence of the colony queen influences the concentration of octopamine, a neuromodulator, in worker ants, which in turn has an effect on nestmate recognition acuity in workers. We demonstrate that queenless workers exhibit reduced brain octopamine levels and reduced discriminatory acuteness; however, feeding queenless workers octopamine restored both. Dopamine levels are influenced by honeybee queen pheromones; however, levels of this biogenic amine were unchanged in our experiments. This is the first demonstration of a link between the presence of the colony queen, a worker biogenic amine, and conspecific nestmate recognition, a powerful expression of colony cohesion and territoriality.