Abstract This study examined the spatial and temporal distribution of serotonin‐immunoreactive (5‐HT‐ir) neurons in the brainstem of Petromyzon marinus at three developmental stages, larval, postmetamorphic, and reproductive. Computer‐assisted 3‐D reconstructions were made of the three main 5‐HT‐ir neuron groups. The rostralmost brainstem group was located near the posterior commissure, the second group at the isthmus, and the third group in the bulbar area. For each of those groups, the distribution of the 5‐HT‐ir neurons was very similar in the three developmental stages examined, suggesting that the 5‐HT system is relatively mature early in larval animals. The soma of 5‐HT‐ir neurons increased in size and their dendritic fields increased in complexity with development. Furthermore, the number of 5‐HT‐ir neurons in each group increased significantly from the larval to the reproductive stage. To determine whether this was due to the genesis of 5‐HT neurons, bromodeoxyuridine (BrdU) was injected into larval, metamorphosing, and postmetamorphic lampreys. These experiments revealed a few neurons colocalizing BrdU and 5‐HT in metamorphosing animals. Taken together, the present results suggest that 5‐HT neurons increase in number during maturation and that neurogenesis could, at least partially, contribute to the appearance of new 5‐HT cells at different developmental stages. J. Comp. Neurol. 495:788–800, 2006. © 2006 Wiley‐Liss, Inc.