The disruptive effect of excessive serotonin (5-HT) levels on the development of cortical sensory maps is mediated by 5-HT1B receptors, as shown in barrelless monoamine oxidase A knock-out mice, in which the additional inactivation of 5-HT1B receptors restores the barrels. However, it is unclear whether 5-HT1B receptors mediate their effect on barrel formation by a trophic action or an activity-dependent effect. To test for a possible effect of 5-HT1B receptors on activity, we studied the influence of 5-HT on the thalamocortical (TC) synaptic transmission in layer IV cortical neurons. In TC slices of postnatal day 5 (P5)-P9 neonate mice, we show that 5-HT reduces monosynaptic TC EPSCs evoked by low-frequency internal capsule stimulation and relieves the short-term depression of the EPSC evoked by high-frequency stimulation. We provide evidence that 5-HT decreases the presynaptic release of glutamate: 5-HT reduces similarly the AMPA-kainate and NMDA components and the paired pulse depression of TC EPSCs. We show also that 5-HT1B receptors mediate exclusively the effect of 5-HT: first, the effect of 5-HT on the TC EPSC is correlated with the transient expression of 5-HT1B receptor mRNAs in the ventrobasal thalamic nucleus during postnatal development; second, it is mimicked by a 5-HT1B agonist; third, 5-HT has no effect in 5-HT1B receptor knock-out mice. Our results show that in the developing barrel field of the neonatal mice, 5-HT1B receptors mediate an activity-dependent regulation of the TC EPSC that could favor the propagation of high-frequency TC activity.