Na+-dependent, fluoxetine-sensitive high-affinity uptake of serotonin and Na+-dependent uptake of glutamate were studied in primary astrocyte cultures from 1-day-old rat neocortex. This uptake was independent of time in culture from 1 to 6 weeks. High-affinity serotonin uptake was decreased when cells were grown in horse serum as compared to fetal bovine serum and was almost absent when cells were grown in chemically defined medium. In contrast, glutamate uptake was unaffected by the composition of the medium in which the cultures were grown. The serum effect on serotonin uptake was not due to the greater level of serotonin in the fetal bovine serum and was only reversed by a change of serum over a time period of days. © 1992 Wiley-Liss, Inc.