Serotonergic activity is influenced by social status and manipulation of social signals. In the lizard Anolis carolinensis, eyespot formation, i.e. darkening of postorbital skin from green to black, appears during stressful and agonistic situations, forming first in males that become dominant. To assess the effect of eyespots on central serotonergic activity during social interaction, males were paired by weight and painted postorbitally with green or black paint. Manipulation of eyespot color influenced social interactions and status. All males that viewed an opponent with black painted eyespots became subordinate. In these subordinate animals, serotonergic activity was elevated in hippocampus, striatum, nucleus accumbens and locus ceruleus. In contrast, males that viewed opponents with hidden eyespots (painted green) and became dominant had increased serotonergic activity in hypothalamus, medial amygdala and raphé. Pre-painted eyespots produced results that distinguish dominant and subordinate relationships based on serotonergic activity not previously seen in unmanipulated pairs. Results from experiments using pairs are similar to those using mirrors for medial amygdala and locus ceruleus, but not hippocampus, nucleus accumbens or raphé. Decreased hypothalamic serotonin was associated with increased aggressive behavior. These results, when compared with previous studies, suggest some flexibility in central serotonergic systems, which may shape dominant and subordinate rank acquisition, and appear to be influenced by the completion of social role formation. Furthermore, social status and central serotonergic activity was influenced by a visual cue, the presence or absence of postorbital eyespots on an opponent.