The pharmacological actions of L-proline on excitatory and inhibitory amino acid receptors have been characterized under voltage-clamp conditions, using cultured dissociated neurons from the dorsal horn of the rat spinal cord. At a holding potential of -62 mV, millimolar concentrations of L-proline elicited an inward current that was partially antagonized by D-(-)-2-amino-5-phosphonopentanoic acid (APV), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and strychnine and was virtually abolished (97% block) by a combination of all three antagonists. Currents evoked by D-proline were abolished by strychnine alone. APV-, CNQX-, and strychnine-sensitive components of L-proline-evoked currents were isolated using various combinations of the three antagonists. These currents were identical to currents elicited by N-methyl-D-aspartate (NMDA), kainate, and glycine, respectively, with respect to antagonist specificity, reversal potential, and ionic permeability. The APV- and strychnine-sensitive currents also showed a time dependence similar to that of the currents elicited by NMDA and glycine. EC50 values could not be calculated, because the response did not saturate within the tested range of L-proline concentrations (0.3-50 mM). Estimates of relative potency were obtained, however, by comparison with responses elicited by selective agonists. The APV-sensitive, CNQX-sensitive, and strychnine-sensitive currents evoked by 10 mM L-proline were comparable in size to currents elicited by 15 microM NMDA, 5 microM kainate, and 30 microM glycine, respectively. L-Proline was found to elicit an increase in intracellular [Ca2+] that was dependent upon Ca2+ entry into the cell. These Ca2+ responses were enhanced by strychnine and partially antagonized by APV, CNQX, or Mg2+. Our results using dorsal horn neurons grown in culture indicate that L-proline is a weak agonist at strychnine-sensitive glycine receptors and at both NMDA and non-NMDA glutamate receptors. These observations should help in interpreting the confusing array of L-proline actions that have been described using more intact nervous system preparations. Furthermore, the ability of L-proline to stimulate Ca2+ entry after activation of excitatory amino acid receptors implicates L-proline as a potential endogenous excitotoxin.