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The multiple partner choice arena (MPCA) is an experimental setup in which male rats display a significant shortening of ejaculation latency, which is the main characteristic of premature ejaculation (PE) in men. Thus, the MPCA is a potential animal model for PE.In this study, we further analyze whether the features of the MPCA satisfy the validity criteria for it to be considered an animal model as well as the possible participation of the serotoninergic system in the faster ejaculation exhibited by male rats in the MPCA.In Experiment 1, male rats were tested in a standard arena to assess their sexual behavior, then were assessed 1 week later in the MPCA. Another group was first tested in the MPCA, then in a standard arena. In Experiment 2, male rats divided into two groups were treated daily with WAY-100635 (5-HT1A antagonist) or vehicle for 15 days. In each group, half of the subjects were tested in a standard arena and half were tested in the MPCA on days 1, 8, and 15 of treatment.Number of intromissions and intromission and ejaculation latencies were the main outcome measures.In Experiment 1, males tested in the MPCA ejaculated significantly faster, regardless of the order in which they were evaluated in both arenas. In Experiment 2, the administration of WAY-100635 increased intromission and ejaculation latencies, and the number of intromissions in the MPCA.The results obtained in the MPCA support its use as an animal model for PE evaluation. Olayo-Lortia J, Ferreira-Nuño A, Velázquez-Moctezuma J, and Morales-Otal A. Further definition on the multiple partner choice arena: A potential animal model for the study of premature ejaculation. J Sex Med 2014;11:2428–2438.