Dorsal root-evoked stimulation of sensory afferents in the hemisected in vitro rat spinal cord produces reflex output, recorded on the ventral roots. Transient spinal 5-HT2Creceptor activation induces a long-lasting facilitation of these reflexes (LLFR) by largely unknown mechanisms. Two Sprague–Dawley substrains were used to characterize network properties involved in this serotonin (5-HT) receptor–mediated reflex plasticity. Serotonin more easily produced LLFR in one substrain and a long-lasting depression of reflexes (LLDR) in the other. Interestingly, LLFR and LLDR were bidirectionally interconvertible using 5-HT2A/2Cand 5-HT1Areceptor agonists, respectively, regardless of substrain. LLFR was predominantly Aβ afferent fiber mediated, consistent with prominent 5-HT2Creceptor expression in the Aβ fiber projection territories (deeper spinal laminae). Reflex facilitation involved an unmasking of polysynaptic pathways and an increased receptive field size. LLFR emerged even when reflexes were evoked three to five times/h, indicating an activity independent induction. Both the NMDA and AMPA/kainate receptor–mediated components of the reflex could be facilitated, and facilitation was dependent on 5-HT receptor activation alone, not on coincident reflex activation in the presence of 5-HT. Selective blockade of GABAAand/or glycine receptors also did not prevent reflex amplification and so are not required for LLFR. Indeed, a more robust response was seen after blockade of spinal inhibition, indicating that inhibitory processes serve to limit reflex amplification. Overall we demonstrate that the serotonergic system has the capacity to induce long-lasting bidirectional changes in reflex strength in a manner that is nonassociative and independent of evoked activity or activation of ionotropic excitatory and inhibitory receptors.