Lalley, Peter M., Olivier Pierrefiche, Anne M. Bischoff, and Diethelm W. Richter. cAMP-dependent protein kinase modulates expiratory neurons in vivo. J. Neurophysiol. 77: 1119–1131, 1997. The adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) second-messenger system influences neuronal excitability by modulating voltage-regulated and transmitter-activated channels. In this study we investigated the influence of the cAMP-PKA system on the excitability of expiratory (E) neurons in the caudal medulla of anesthetized, paralyzed, and artificially ventilated adult cats. We intracellularly injected the PKA inhibitors cAMP-dependent PKA inhibitor 5-22 amide (Walsh inhibitory peptide) and R_p-adenosine 3′,5′-cyclic monophosphothioate triethylamine (R_p-cAMPS), the PKA activator S_p-adenosine 3′,5′-cyclic monophosphothioate triethylamine (S_p-cAMPS), and the adenylyl cyclase activator forskolin and measured membrane potential, neuronal input resistance, and synaptic membrane currents. Inhibition of cAMP-PKA activity by Walsh inhibitory peptide or R_p-cAMPS injections hyperpolarized neurons, decreased input resistance, and depressed spontaneous bursts of action potentials. Action potential duration was shortened and afterhyperpolarizations were increased. Inhibitory synaptic currents increased significantly. Stimulation of cAMP-PKA activity by S_p-cAMPS or forskolin depolarized neurons and increased input resistance. Spontaneous inhibitory synaptic currents were reduced and excitatory synaptic currents were increased. R_p-cAMPS depressed stimulus-evoked excitatory postsynaptic potentials and currents, whereas S_p-cAMPS increased them. S_p-cAMPS also blocked postsynaptic inhibition of E neurons by 8-hydroxy-dipropylaminotetralin, a serotonin-1A (5-HT-1A) receptor agonist that depresses neuronal cAMP-PKA activity. To determine the predominant effect of G protein-mediated neuromodulation of E neurons, we injected guanosine-5′-O-(3-thiotriphosphate) tetralithium salt (GTP-γ-S), an activator of both stimulatory and inhibitory G proteins. GTP-γ-S hyperpolarized E neurons, reduced input resistance, and increased action potential afterhyperpolarization. We conclude that the intracellular cAMP-PKA messenger system plays an important role in the activity-dependent modulation of excitability in E neurons of the caudal medulla. In addition, the cAMP-PKA pathway itself is downregulated during activation of 5-HT-1A receptors.