The neurotrophin brain-derived neurotrophic factor (BDNF) was tested for its ability to promote the survival and regulation of expression of phenotypic markers of dopaminergic, serotonergic, and GABAergic neurons in free-floating roller tube cultures of human fetal ventral mesencephalon. This culture system contains neurons of the anlage of the substantia nigra as well as that of the rostral raphe nucleus. Dopaminergic neuron number and tyrosine hydroxylase (TH) fiber density was monitored by TH immunocytochemistry. Measurement of dopamine (DA) content, TH enzymatic activity, serotonin (5-HT) content, and glutamic acid decarboxylase (GAD) activity were used as indices of their respective neurotransmitter function. The presence of GABAergic and serotonergic neurons in this culture system was confirmed by GABA and 5-HT immunocytochemistry. In cultures maintained in the presence of BDNF (10 ng/ml), the density of TH-positive cells was increased by 2.5-fold (P F 0.05), and the TH-positive fiber density was increased by 3.5-fold (P F 0.01), relative to control cultures. Similarly, the relative increases in DA content and TH activity were 2.6- and 2.3-fold, respectively, in the BDNF-treated cultures (P F 0.01 and P F 0.01). On a per neuron basis, DA content and TH activity were not markedly changed by BDNF treatment, suggesting that the increases in DA content and TH activity are due to more DA neurons surviving. Relative elevations were also observed in serotonin content (2.0-fold, P F 0.01) and GAD enzymatic activity (1.4-fold, P F 0.01). Future studies will need to determine whether these changes result from the direct action of BDNF on these neurons or through some indirect mechanism. The results demonstrate that BDNF has beneficial effects on cultured human fetal tissue, which may be relevant in optimizing neuronal transplantation techniques, and that multiple systems are simultaneously influenced by BDNF.