The high affinity L-proline transporter (PROT) is a member of the family of Na+ (and Cl-)-dependent plasma membrane transport proteins that comprises transporters for several neurotransmitters, osmolytes, and metabolites. The brain-specific expression of PROT in a subset of putative glutamatergic pathways implies a specialized function for this novel transporter and its presumed natural substrate L-proline in excitatory synaptic transmission. However, definitive studies of the physiological role(s) of high affinity L-proline uptake have been precluded by the lack of specific uptake inhibitors. Here, we report that Leu- and Met-enkephalin and their des-tyrosyl derivatives potently and selectively inhibited high affinity L-proline uptake in rat hippocampal synaptosomes and in PROT-transfected HeLa cells. High concentrations of the opiate receptor antagonist naltrexone did not block the inhibitory actions of these peptides, arguing against an involvement of opioid receptors. Des-tyrosyl-Leu-enkephalin elevated the apparent K(m) of L-proline transport in transfected HeLa cells without altering the V(max). PROT-transfected HeLa cells did not accumulate [3H]Leu-enkephalin above background levels, demonstrating that enkephalins are not substrates for PROT. These findings indicate that enkephalins competitively inhibit mammalian brain PROT through a direct interaction with the transporter protein at or near the L-proline binding site. The high potency and specificity of des-tyrosyl-Leu-enkephalin make this compound a useful tool for elucidating the structure-function properties and physiological role(s) of PROT.