We were interested in the changes spinal locomotor networks undergo during development. The zebrafish embryos and larvae are important model systems for the development of the vertebrate nervous system, but there is little knowledge about the maturation of the locomotor circuitry as the animal grows into adulthood. Because of this, we developed an in vitro preparation of the isolated spinal cord where we could induce fictive locomotion pharmacologically or through electrical stimulation of descending axons. We recorded from spinal interneurons and motoneurons using the patch-clamp technique and analyzed their morphology in a confocal laser scanning microscope. We investigated the action of an important neuromodulator, serotonin, on the locomotor network and found that its actions are quite different in the adult to what has been shown in the larva. In our preparation, serotonin is endogenously released and decreases the locomotor frequency by increasing the inhibitory currents the network neurons receive during every cycle of rhythmic activity. Also, we investigated the recruitment of motoneurons during swimming at different frequencies and could show that, again unlike in the larva, it is not in accordance with the classical ‘size principle’. Instead we found that motoneurons can be divided into four groups with distinct locations in the spinal cord that receive different synaptic drive from premotor interneurons. Interestingly, we could also show that these motoneuron populations possess different intrinsic properties that contribute to their firing behavior.