Neurotransmitters and their receptors have been involved in both proper brain development and neurodevelopmental disorders. The role that nicotinic receptors play in immature cortical neurons was initially investigated by gene profiling using Affymetrix DNA arrays. Both short (15 min) and prolonged (18 h) treatments with nicotine did not induce modification in gene expression, whereas a significant down-regulation of c-fos protein levels was observed after 18 h treatment. Conversely, a brief treatment with the glutamatergic agonist NMDA triggered up-regulation of immediate early genes and transcription factors, which remained unaffected by pre-treatment for 18 h with nicotine. Calcium imaging studies revealed that NMDA activated a sustained increase in intracellular calcium concentration in the majority of neurons, whereas nicotine evoked only a transient calcium increase in a smaller percentage of neurons, suggesting that the calcium signalling response was correlated with activation of gene expression. Nicotine effects on immature cortical neurons perhaps do not require gene regulation but may be still acting on signalling pathways.