The cAMP-protein kinase A (PKA) pathway plays a critical role in regulating neuronal activity. Yet, how PKA signalling shapes the population activity of neurons that regulate respiratory rhythm and motor patterns in vivo is poorly defined. We determined the respiratory effects of focally inhibiting endogenous PKA activity in defined classes of respiratory neurons in the ventrolateral medulla and spinal cord by microinjection of the membrane-permeable PKA inhibitor Rp-adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS) in urethane-anaesthetized adult Sprague Dawley rats. Phrenic nerve activity, end-tidal CO2 and arterial pressure were recorded. Rp-cAMPS in the preBötzinger complex (preBötC) caused powerful, dose-dependent depression of phrenic burst amplitude and inspiratory period. Rp-cAMPS powerfully depressed burst amplitude in the phrenic premotor nucleus, but had no effect at the phrenic motor nucleus, suggesting a lack of persistent PKA activity here. Surprisingly, inhibition of PKA activity in the preBötC increased phrenic burst frequency, whereas in the Bötzinger complex phrenic frequency decreased. Pretreating the preBötC with strychnine, but not bicuculline, blocked the Rp-cAMPS-evoked increase in frequency, but not the depression of phrenic burst amplitude. We conclude that endogenous PKA activity in excitatory inspiratory preBötzinger neurons and phrenic premotor neurons, but not motor neurons, regulates network inspiratory drive currents that underpin the intensity of phrenic nerve discharge. We show that inhibition of PKA activity reduces tonic glycinergic transmission that normally restrains the frequency of rhythmic respiratory activity. Finally, we suggest that the maintenance of the respiratory rhythm in vivo is not dependent on endogenous cAMP-PKA signalling.