Reuptake of neuroactive amino acids by high affinity transport systems (Km approximately 10 micrometer) is thought to terminate the action of these substances. Since homoexchange can complicate the interpretation of uptake experiments, it is necessary to demonstrate net inward transport of neuroactive amino acids before uptake can be considered as a likely mechanism for transmitter inactivation. When rat cortical synaptosomes are incubated with 10 micrometer [14C]L-aspartate, net (chemical) and apparent (radioactive) uptake into the synaptosomal fractions are equivalent. Although there is net aspartate uptake into the synaptosome fraction, aspartate exchange can be demonstrated in a variety of conditions. Net uptake exhibits the characteristics of high-affinity transport systems including Na+-and temperature-dependence. Furthermore, KCl (or RbCl)--1 micrometer--are required for net uptake but not radioactive or apparent uptake. LiCl, NH4Cl, CsCl, and choline chloride fail to support net uptake. Ouabain (0.1 micrometer) inhibits net uptake to a greater extent than apparent uptake. Although glutamate inhibits aspartate uptake (and vice versa), the net uptake of the combination is greater than that of each alone. The demonstration of net uptake of aspartate by a high-affinity system is consonant with the idea that this system may play a role in its inactivation in the synaptic region.