Respiration-related membrane potential fluctuations were recorded in hypoglossal (XII) motoneurons and pre-Bötzinger complex (pre-BötC) interneurons in medullary slices from perinatal rats. Bath application of serotonin (5-HT) evoked a ketanserine-sensitive depolarization (approximately 11 mV) and tonic spike discharge in XII motoneurons, whereas pre-BötC neurons responded with a <6 mV depolarization and no tonic discharge. The membrane effects were accompanied by an increase in respiratory frequency by up to 260% in 64% of preparations. A frequency decrease leading to block of respiratory activity could also occur (20%) as well as an initial acceleration that turned into a frequency depression (16%). In contrast, iontophoresis of 5-HT into the pre-BötC exclusively increased respiratory frequency by 30-220%, whereas iontophoresis into the XII nucleus did not change respiratory frequency but induced tonic nerve discharge. The effects of local iontophoretic administration of 5-HT on membrane properties of XII and pre-BötC cells were very similar to those upon bath application. Bath application and iontophoresis of the 5-HT2 receptor agonist -methyl-hydroxytryptamine mimicked the effects of 5-HT. Bath application of the 5-HT1A receptor agonist 8-hydroxydipropylaminotetralin hydrobromide did not affect XII nerve bursting or pre-BötC neurons. Iontophoresis of 8-hydroxydipropylaminotetralin hydrobromide had almost no effect on respiratory frequency and induced in the interneurons either a depolarization or hyperpolarization (<5 mV) which was blocked by the 5-HT1A receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)N-2-pyridinylcyclohexane carboxamide. In conclusion, 5-HT-evoked tonic excitation of respiratory XII motoneurons is mediated by postsynaptic 5-HT2 receptors. The excitatory effects on respiratory rhythm are also primarily attributable to postsynaptic 5-HT2 receptors of pre-BötC neurons. Additional modulatory effects on the interneurons appear to be mediated by postsynaptic 5-HT1A receptors.