In male hamsters, puberty is associated with increased serotonin innervation and unusual responses to fluoxetine, such as enhanced play-fighting activity against intruders but also an acceleration of its maturation from attacks focused on the face (frontal attacks) to the lower belly and rump, suggesting a role for serotonin (5-HT). We tested the role of 5-HT 1A and 5-HT 3 receptor subtypes on play-fighting behavior observed during resident intruder tests through peripheral treatment with receptor agonists and antagonists. Contrary to observations in adult hamsters, we did not observe any overarching effects of treatment on measures of play-fighting activity, nor its maturation from frontal attacks. However, secondary analyses highlighted variability within the datasets. A subgroup of animals presented inhibited play-fighting activity in response to treatment with DPAT, a 5-HT 1A receptor agonist, but these animals also showed enhanced locomotor activity and reduced interest in engaging their opponents. In addition, early juvenile agonistic behavior was predictive of responsiveness to other treatments. The 5-HT 1A receptor antagonist, WAY, caused a reduction in play-fighting activity in high attackers and an increase in low attackers. Though high attackers under pretest conditions were equally inhibited by CBG, a 5-HT 3 receptor agonist, they performed a higher proportion of frontal attacks. Finally, the density of 5-HT 1A and 5-HT 3 receptor immunoreactivity was compared among subjects sampled at postnatal Day 35 (early puberty) or postnatal Day 70 (adulthood) within areas mediating the control of social behavior in adults. Adult males showed a higher density of immunolabeling for 5-HT 1A receptors in the anterior hypothalamus and medial amygdala, as well as 5-HT 3 receptors in the lateral septum. The data suggest that the development of 5-HT receptor expression participates in the control of play-fighting activity and its maturation during puberty in male hamsters., (© 2025 Wiley Periodicals LLC.)