Abstract Receptor subtypes involved in the 5-hydroxytryptamine (5-HT)-induced depression of synaptic transmission in neonatal rat spinal cords in vitro were evaluated in the absence or presence of Mg2+ in the medium. Stimulation of a dorsal root evoked monosynaptic reflex potential (MSP) and polysynaptic reflex potential (PSP) in the segmental ventral root in Mg2+-free medium where the voltage-dependent blockade of NMDAreceptors is absent. The 5-HT (0.3–50μM) in the Mg2+-free medium depressed the MSPand PSP in a concentration-dependent manner. At 30μM of 5-HT, the depression was 57% and 95% for MSP and PSP, respectively, and no further depression was seen at 50μM. The 5-HT-induced depression of the reflexes in the Mg2+-free medium was blocked by ondansetron (5-HT3 receptor antagonist), but not by spiperone (5-HT2A/2C antagonist). In the Mg2+-free medium, phenylbiguanide (5-HT3 agonist) also depressed the MSPand PSP in a concentration-dependent manner and was blocked by ondansetron. Addition of Mg2+ (1.3mM) to the medium abolished the PSP and decreased the MSPby 30%. In the presence of Mg2+, 5-HT (1–50μM) also depressed the MSP in a concentration-dependent manner. At 10μM of 5-HT, there was approximately 20% depression and at 50μM the depression was 100%. The 5-HT-induced depression of MSP in the Mg2+-containing medium was antagonized by spiperone (p<0.05, two-way ANOVA), but not by ondansetron. The results indicate that the 5-HT-induced depression of MSPinvolves 5-HT3 receptors in the Mg2+-free medium and 5-HT2A/2C in the presence of Mg2+ when NMDA receptors are in the closed state.