The uptake of L-proline by synaptosomes isolated from different regions of the brain was investigated. The highest rates of transport and the largest accumulation ratios were found in synaptosomes from the midbrain, striatum, hippocampus and hypothalamus, with lower activity in the cortex and medulla (+ pons) and lowest in the cerebellum. The high affinity, Na+-dependent proline uptake had a Km of 12 microM, a Vmax of 0.6 nmol . min-1 . mg protein-1 and the maximum accumulation ratio ( [proline]i/[proline]o) at 2 microM added radioactive amino acid was 40-50. Our results suggest that: (1) only 5-10% of the synaptosomal population accumulates proline through the high-affinity uptake; (2) the characteristics of the proline uptake system into the prolinergic nerve endings are remarkably similar to those of other amino acid neurotransmitters (GABA, aspartate and glutamate); (3) the behavior of proline is consistent with this amino acid being a neurotransmitter in the central nervous system.