The establishment of cortical synaptic circuits during early development requires the presence of subplate neurons (SPn), a heterogeneous population of neurons capable to integrate and process synaptic information from the thalamus, cortical plate and neighboring SPn. An accumulation of cholinergic afferents and nicotinic acetylcholine receptors (nAChR) has been documentated in the subplate around birth. To assess the developmental role of the cholinergic innervation onto SPn, we used whole-cell patch-clamp recordings of visually identified and biocytin-labeled SPn in neonatal rat somatosensory cortex. Functional nAChR were present in 92% of the investigated SPn. Activation of postsynaptic nAChR by local application of agonists elicited a brief membrane depolarization associated with a barrage of action potentials and large inward currents reversing around 0 mV. According to our pharmacological data excitation of SPn is mediated by 42 receptors. In contrast, functional 7 nAChR could not be identified on SPn. Activation of nAChR affected neither the spontaneous synaptic activity of SPn nor the synaptic connections between thalamus and SPn and within subplate. Nicotine, at concentrations reaching the developing brain by maternal smoking, induced a severe desensitization of nAChR and an increase in the baseline noise. These results indicate that nAChR-mediated excitation of SPn may stabilize the developing synaptic circuits and suggest the involvement of nAChR located on SPn in the fetal tobacco syndrome.