The effects of S-100B, nerve growth factor (NGF), epidermal growth factor (EGF) and insulin were tested in cultured mesencephalic neurons. Only chronic S-100B showed enhancement (maximal at 3.2 ng/ml is 171%) after 3 days of incubation of the [3H]5-HT uptake capacity by serotonergic neurons. A single application at initial plating of S-100B (maximal at 5 ng/ml is 185%), but not calmodulin, increased the development of the [3H]5-HT uptake capacity by the cultured serotonergic neurons. Morphometric analysis of cultured 5-HT immunoreactive (IR) neurons showed an increase (135 and 147%) in neurite length 30 h after S-100B application of 16 and 3.2 ng/ml (respectively). These results suggest that S-100B is a serotonergic growth factor in the mammalian brain.