Besides their neurotransmitter and/or neuromodulatory roles, many neuroactive substances synthesized and released during brain development can also directly influence neuronal differentiation. Transitory expression of neurotransmitters, their metabolic enzymes and their receptors is only one aspect of this trophic role. The most considerable progress in neurotrophic factor research has been made with the use of primary cultures of neuronal cells, and numerous studies have focused on the effects of neurotransmitters on the differentiation of cells at various stages of development. Thus, several neuropeptides like VIP, substance P, enkephalins, somatostatin, and monoamines, can modulate neuronal differentiation, but only during a limited period of fetal life. Among the monoamines, it was shown that, depending on the target, 5-HT stimulates the development of the neuropile, the myelinization of axons, the differentiation of the synaptic contacts, induces markers of monoaminergic neuron differentiation, inhibits the development of the growth cone, decreases the branching of neurites, and influences the survival, cell body size, and neurite outgrowth in several neuronal cultures. 5-HT can also indirectly influence the differentiation of serotonergic neurons by the intermediate of astrocytes, and it was shown in our laboratory that 5-HT1A agonists can stimulate the cholinergic parameters of primary cultures of rat fetal septal neurons. At the molecular level, the events triggered by neurotransmitters that underlie their neurotrophic action probably involve the transmembrane influx of calcium. To date, calcium regulation of cellular processes is one of the most rapidly expanding areas of research in developmental neurobiology.