Glycinergic neurotransmission was examined in rat medial pontine reticular formation neurons in vitro. Intracellular recordings using glass microelectrodes were made in acutely prepared brainstem slices 400 microns thick. Spontaneous and electrically evoked synaptic activity was blocked by the glycine antagonist strychnine (1-5 microM) but not by the GABA antagonists bicuculline methiodide (40 microM) or picrotoxin (40 microM). Strychnine-sensitive spontaneous and evoked postsynaptic potentials persisted in the presence of the glutamate antagonist (kynurenate, 1 mM). Whole-cell voltage-clamp recordings were carried out in organotypic cultures of rat brainstem. The reversal potential of synaptic currents and responses to exogenously applied glycine were similar and were sensitive to manipulations of the chloride equilibrium potential. Synaptic activity but not responses to exogenous glycine were blocked by tetrodotoxin (0.3 microM). These results indicate the presence of robust, chloride ion-mediated glycinergic inhibition of medial pontine reticular formation neurons, and suggest that glycinergic neurons play an important role in controlling pontine premotor circuitry.