The known neurochemical effect of most currently available antidepressants is the enhancement of the synaptic levels of monoamine neurotransmitters. However, the existence of other mechanisms has been suggested to justify the significant delay between the modulation of the monoaminergic system and the clinical effects. In order to investigate the effects of the antidepressant fluoxetine (a prototypical serotonin selective re-uptake inhibitor) and to improve the understanding of its mechanism of action, we performed a proteomic investigation in rat primary cortical neurons exposed sub-chronically to this antidepressant. Cortical neurons were treated for 3 days with 1 microM fluoxetine or vehicle. Protein extracts were processed for 2D gel characterization. Image analysis allowed the identification of six proteins differently expressed by more than 100% and seven proteins differently expressed by more than 50% (P<0.05). Nine proteins were identified by mass spectrometry. Among them, cyclophilin A, 14-3-3 protein z/delta and GRP78 are involved in neuroprotection, in serotonin biosynthesis and in axonal transport, respectively. This study showed that the primary culture of cortical neurons is a suitable system for studying the effects of fluoxetine action and may contribute to improve the understanding of fluoxetine psychotherapeutic action and the mechanisms mediating the long-term effects of this antidepressant treatment.