Isolated spinal cords prepared from neonatal rats were used to examine the effects of l‐proline (l‐Pro). l‐Pro (1–8 mm) depolarized ventral and dorsal roots in a dose‐dependent manner with one sixth of the potency of l‐glutamate (l‐Glu). l‐Pro was four times more potent than d‐Pro. Prolonged application of l‐Pro produced a plateau depolarization of motoneurones with no apparent fade. Omission of calcium ions from the medium potentiated the depolarizing actions of l‐Pro, l‐Glu and quisqualate. L‐Pro was antagonized by concentrations of 2‐amino‐5‐phosphonovalerate (25 μm), γ‐d‐glutamylglycine (100 μm) and Mg2+ ions (1 mm) that depressed responses to N‐methyl‐d‐aspartate (NMDA). The NMDA receptor‐mediated component of the response to l‐Pro was estimated to be 60–70%. These data suggest that l‐Pro should be considered as a possible excitatory neurotransmitter and that, because l‐Pro is a neutral compound, excitatory amino acid receptors may not require an agonist to possess two anionic groups and one cationic group.