Serotonergic modulation of ion channels in the dendrites of facial motoneurones (FMs) can markedly influence the integration of EPSPs. EPSPs may also be subject to modulation through mechanisms that influence glutamate release. We have used whole-cell, patch-clamp recordings and neonatal rat brainstem slices, prepared from humanely killed 4 to 14-day-old rats, to characterise excitatory glutamatergic transmission onto FMs and investigate its modulation by serotonin (5-HT). Effects were statistically significant (P≤0.05) using an unpaired Student’s t test, unless otherwise stated. Minimal stimulation with an electrode placed close to the dendrites of a FM evoked EPSCs that fluctuated in amplitude and occasionally failed to generate a response. At a holding potential of −70 mV, EPSCs had mean amplitudes, rise and decay time constants of −80 ± 33 pA, 1.8 ± 0.6 and 11.1 ± 3.0 ms, respectively (n = 12, mean ± S.D.). EPSC current-voltage relationships displayed outward rectification with a V_rev of −17 ± 11 mV (n = 4). Application of NBQX (20 μM) reduced mean EPSC amplitude to 16 ± 11 % of control (n = 12). In the presence of NBQX, holding at +20 mV revealed an EPSC with a mean amplitude of 83 ± 20 pA while at −70 mV removal of external Mg²⁺ uncovered an EPSC −42 ± 16 pA in amplitude. Both EPSCs were inhibited by AP-5 (50 μM, n = 4). EPSCs displayed paired pulse facilitation which when averaged after excluding failures occasionally resulted in paired pulse EPSCs of equal amplitude suggesting stimulation of a single release site. Bath-application of either 5-HT (10 μM) or the 5-HT_1B-receptor agonist, CP93129 (10 μM), significantly reduced the mean EPSC amplitude at −70 mV to 31 ± 26 % (n = 15) and 20 ± 15 % (n = 4) of control, respectively (one sample Student’s t test). The 5-HT_1A-receptor agonist, 8-OH-DPAT (10 μM), failed to significantly alter EPSC amplitude (85 ± 14 %, n = 3, one sample Student’s t test). Application of the 5-HT_1B-receptor antagonist, isamoltane (1 μM), reduced the 5-HT-mediated inhibition of EPSC amplitude from 26 ± 15 % to 49 ± 18 % (n = 4), whereas the 5-HT_1A-receptor antagonist, WAY-100635 (1 μM), had no effect on the response to 5-HT (20 ± 13 % of control, n = 3). The reduction in EPSC amplitude was associated with a significant increase in the EPSC failure rate from 25 ± 19 % to 65 ± 27 % for 5-HT (n = 15) and 26 ± 25 % to 88 ± 14 % for CP93129 (n = 4). Examination of miniature EPSCs in the presence of TTX (0.3 μM) showed a reduction in frequency from 3.1 ± 1.6 to 2 ± 1.2 Hz in the presence of 5-HT, while amplitude was not significantly different (69 ± 19 to 55 ± 11 pA) (n = 3, Kolmogorov-Smirnov two sample test). In summary, both non-NMDA and NMDA receptors contribute to excitatory synaptic transmission in the facial nucleus. Our data indicate that in addition to altering the integrative properties of FMs, 5-HT also regulates excitatory synaptic transmission through mechanisms that modulate glutamate release.