1. Spontaneous motoneuronal activity was monitored from the lumbar ventral roots in an isolated spinal cord preparation from rat fetuses at embryonic days (E) 13.5-18.5. 2. Spontaneous bursts that were synchronized in both left and right ventral roots were observed periodically (mean interval, 1.5-2.6 min) from E14.5 to 17.5. This activity was abolished in Ca(2+)-free saline or by application of tetrodotoxin (1 microM), indicating that it was synaptically mediated. 3. The glutamate receptor blocker kynurenate (4 mM) failed to block spontaneous bursts at E14.5-15.5, though it completely abolished them at E17.5. The glycine receptor antagonist strychnine (10 microM) completely blocked spontaneous bursts at E14.5-15.5. Bicuculline, a GABAA receptor antagonist, reduced the amplitude of the spontaneous bursts. 4. At E15.5, a brief application of glycine (250 microM to 2 mM) evoked excitatory responses resembling the spontaneous bursts in both time course and amplitude. Such glycine-induced responses were not observed under Ca(2+)-free conditions, suggesting that they were synaptically evoked. These synaptic responses were not blocked by kynurenate (4 mM), but they were abolished by strychnine (10 microM). 5. It is concluded that glycine and GABA generate the earliest spontaneous motor activity of the fetus and function transiently as excitatory transmitters in the embryonic spinal cord.