Dissociated mesencephalic raphe cells from fetal rats (14-18 days) were grown in culture in 96 well Linbro plates. The maturation of serotonergic cells was qualitatively studied using immunocytochemistry with a serotonin antibody and quantitatively by measuring the retention of radioactivity following incubation in the presence of a low concentration of [3H]5-hydroxytryptamine (6 X 10(-8) M). The 5-hydroxytryptamine immunoreactive neurons showed specific staining in the perikaryon, nucleus, dendrites, axons and growth cones. These neurons formed varicose fibers and growth cones after 18 h in culture and survived for up to 21 days in culture. Each serotonergic neuron concentrated approximately 1 fmol of serotonin after 20 min of incubation. Maturation of mesencephalic serotonergic neurons was increased in co-cultures of both normal (hippocampus, cerebral cortex, olfactory bulb and striatum) and abnormal (spinal cord) target neurons. The best stimulation was produced by dissociated hippocampal neurons (14-18 days of gestation) on mesencephalic raphe cells (14 days of gestation) after 4 days in culture. This stimulation was seen in culture conditions which favored neuronal but not glial survival. Our results obtained using cultures of dissociated serotonergic cells are consistent with an expansive network pattern developed by this chemical transmitter system in the adult brain.