Gramicidin-perforated patch-clamp recording revealed phasic Cl(-)-mediated hyperpolarizations in respiratory neurons of the brainstem-spinal cord preparation from newborn rats. The in vitro respiratory rhythm persisted after block of gamma-aminobutyric acid (GABA), i.e. GABAA, receptor-mediated inhibitory postsynaptic potentials (IPSPs) with bicuculline and/or glycinergic IPSPs with strychnine. In one class of expiratory neurons, bicuculline unmasked inspiration-related excitatory postsynaptic potentials (EPSPs), leading to spike discharge. Bicuculline also blocked hyperpolarizations and respiratory arrest due to bath-applied muscimol, whereas strychnine antagonized similar responses to glycine. The reversal potential of respiration-related IPSPs and responses to GABA, muscimol or glycine was not affected by CO2/HCO3(-)-free solutions, but shifted from about -65 mV to values more positive than -20 mV upon dialysis of the cells with 144 instead of 4 mM Cl-. Impairment of GABA uptake with nipecotic acid or glycine uptake with sarcosine evoked a bicuculline- or strychnine-sensitive decrease of respiratory frequency which could lead to respiratory arrest. Also, the GABAB receptor agonist baclofen led to reversible suppression of respiratory rhythm. This in vitro apnoea was accompanied by a K+ channel-mediated hyperpolarization (reversal potential -88 mV) of tonic cells, whereas membrane potential of neighbouring respiratory neurons remained almost unaffected. Both baclofen-induced hyperpolarization and respiratory depression were antagonised by 2-OH-saclofen, which did not affect respiration-related IPSPs per se. The results show that synaptic inhibition is not essential for rhythmogenesis in the isolated neonatal respiratory network, although (endogenous) GABA and glycine have a strong modulatory action. Hyperpolarizing IPSPs mediated by GABAA and glycine receptors provide a characteristic pattern of membrane potential oscillations in respiratory neurons, whereas GABAB receptors rather appear to be a feature of non-respiratory neurons, possibly providing excitatory drive to the network.