Endogenous amines and peptides continuously modulate the activity of neuronal networks and are required even for their normal operation. The respiratory rhythm generator, localized in the pre-Bötzinger complex, is not an exception. This network is modulated by various neurotransmitters, including serotonin (5-HT). In this study, we isolated the respiratory network in brainstem slices and demonstrate that the endogenous activation of 5-HT(2A) is required for the generation of the respiratory rhythm in vitro. At the network level, activation of 5-HT(2A) receptors with 4-iodo-2,5-dimethoxyamphetamine or the 5-HT uptake blocker alaproclate increased the frequency of respiratory activity. Blockade of endogenously activated 5-HT(2A) receptors with three different antagonists decreased the frequency, amplitude, and regularity of respiratory population activity, an effect that was blocked by protein kinase C (PKC) activators. At the cellular level, blockade of 5-HT(2A) receptors reduced the action potential discharge in all examined respiratory neurons, which was associated with a reduction in the fast and the persistent sodium current. Continuous application of 5-HT(2A)-receptor antagonists differentially affected pacemaker neurons. Pacemaker activity was eliminated in cadmium-insensitive pacemaker neurons. In cadmium-sensitive pacemaker neurons, the frequency of pacemaker activity was unaffected and the amplitude of pacemaker bursts was enhanced. It is assumed that cadmium-insensitive pacemakers rely on the persistent sodium current, whereas cadmium-sensitive pacemakers depend on the activation of calcium currents. We conclude that endogenously activated 5-HT(2A) receptors are required for maintaining fictive respiratory activity in the brainstem slice by modulating sodium conductances via a PKC pathway.