The amino acid proline has long been suspected to serve as a modulator of synaptic transmission in the mammalian brain, but no such function has been identified. The selective expression of high affinity proline transport by a subset of glutamate pathways suggested that proline might play a role in synaptic transmission at these sites. This idea was tested with use of one such pathway, the Schaffer collateral-commissural projection to CA1 pyramidal cells of the rat hippocampus. Proline enhanced the initial slope of the field EPSP without affecting axonal excitability or the magnitude of paired-pulse facilitation. Proline-induced potentiation far outlasted the period of proline application and required the activation of NMDA receptors. Proline enhanced Schaffer collateral-commissural synaptic transmission even when the connections between areas CA1 and CA3 had been interrupted. Potentiation was observed with a proline concentration normally present in human CSF (3 microM). A concentration typical of CSF in persons with the genetic disorder hyperprolinemia type II (30 microM) produced a somewhat greater effect. Occlusion experiments suggested that proline-induced potentiation and tetanus-induced long-term potentiation utilize largely distinct transduction mechanisms. Proline-induced potentiation could be blocked by a prior high frequency stimulus, whether or not the stimulus evoked long-term potentiation. These results suggest that endogenous extracellular proline regulates the basal function of some glutamate synapses by maintaining them in a partially potentiated state. They may also facilitate understanding of the seizures and/or mental retardation associated with genetic disorders of proline metabolism.