The characteristics of serotonin-induced potentials change during normal development of spinal cords of rat embryos. These changes are temporally correlated with the growth of serotonin projections into the spinal cord. To determine whether serotonin (5-hydroxytryptamine, 5-HT) in the growing projections is responsible for modulating the changes in neuronal responses to 5-HT, 5-HT synthesis was blocked, and motoneuron responses to exogenous 5-HT were studied 1-2 weeks later. Starting at Day 9 of gestation, prior to the generation of 5-HT neurons in the medulla, pregnant rats were injected daily with p-chlorophenylalanine (p-CPA) which suppressed 5-HT synthesis. p-CPA was effective in reducing 5-HT concentrations because immunoreactive 5-HT projections were absent in embryonic spinal cords of p-CPA-treated rats. However, suppression of 5-HT synthesis did not affect the onset and time course of development of 5-HT-induced potentials. Furthermore, in the absence of 5-HT, the potentials generated by 5-HT were significantly larger than those produced in motoneurons of control rats. These findings indicated that the number of receptors or their binding affinity increased in the absence of 5-HT. The increased responses in p-CPA-treated rats were mediated via 5-HT2/5-HT1C receptors, while activation of other 5-HT1 receptor subtypes and 5-HT3 receptors induced similar depolarizations in p-CPA-treated and untreated rats. Our study suggested that 5-HT was not required for the onset of receptor expression in developing spinal neurons, but it may be responsible for receptor down-regulation.