Monogamous prairie voles (Microtus ochrogaster) show mating-induced aggression towards conspecific strangers. This behavior is both selective and enduring. The present study was designed to investigate the behavioral conditions for the emergence of selective aggression (by varying prior experience with a female and identity of intruders) and the limbic activation in response to an intruder (by mapping regional staining for c-fos) in male prairie voles. In a first experiment, males that mated with a female for 24 h exhibited aggression towards a male intruder and had more Fos-immunoreactive (Fos-ir) cells in the medial amygdala (AMYGme) and medial preoptic area (MPO) relative to males that cohabited with a female without mating or that had no prior exposure to a female. Cohabited males did not become aggressive. However, these males along with mated males had an increased number of Fos-ir cells in the lateral septum (LS) and the bed nucleus of the stria terminalis (BST) relative to males without prior exposure to a female. In a second experiment, mated males exhibited more offensive aggression to a male intruder but more defensive aggression to a female intruder. Both types of aggression, however, induced an increase in the number of Fos-ir cells in the AMYGme. In addition, Fos-ir staining in the BST was induced selectively in response to a male intruder and a similar trend was found in the LS. Exposure to a male or female intruder did not increase Fos-ir staining in the MPO. Taken together, our data suggest the neural substrates activated by social/sexual activity and involved in response to intruders. The AMYGme was involved in processing intruder-related cues and/or in the regulation of aggressive response to both male and female intruders. The BST and LS were modulated by social experience with a female (mating or cohabitation) and were responsive to male-related cues even in the absence of aggression. Finally, the MPO was activated at different magnitudes by social or sexual experience but did not respond to intruder-related cues as measured by Fos-ir.