Monogyne fire ant, Solenopsis invicta, colony workers are territorial and are aggressive toward members of other fire ant colonies. In contrast polygyne colony workers are not aggressive toward non-nestmates, presumably due to broader exposure to heritable and environmentally derived nestmate recognition cues (broad template). Workers from both monogyne and polygyne fire ant colonies execute newly mated queens after mating flights. We discovered that after removal of their colony queen, monogyne worker aggression toward non-nestmate conspecifics quickly drops to investigative levels; however, heterospecific recognition/aggression remains high. Queenless monogyne or polygyne worker groups were also not aggressive toward newly mated queens. Queenless worker groups of both forms that adopted a monogyne-derived newly mated queen became aggressive toward non-nestmate workers and newly mated queens. We suggested that this powerful effect of queens on conspecific nestmate recognition is caused by a queen produced recognition primer pheromone that increases the sensitivity of workers to subtle quantitative differences in nestmate recognition cues. Biogenic amines have been reported to modulate the sensitivity of insects to stimuli. We used this information to probe the primer pheromone/endocrine basis of nestmate recognition in fire ants. Queenright colonies were divided into three components: queenright, queenless, and queenless fed the biogenic amine, octopamine (OA). Queenright colonies maintained high aggression levels. In contrast, queenless workers fed only crickets and aqueous sucrose had low aggression levels. Workers that were fed octopamine had aggression levels that were not significantly different from queenright workers. Feeding OA to fire ant workers was adequate to simulate the presence of the queen, in terms of nestmate recognition. Thus, we have strong evidence that the queen recognition primer pheromone acts on workers to maintain high levels of OA that up - modulates worker sensitivity to the subtle changes in intraspecific nestmate recognition cues.