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-HT_2C receptor activation induces a long-lasting facilitation of these reflexes (LLFR) by largely unknown mechanisms (Machacek et al., 2001). 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 bi-directionally interconvertible using 5-HT_2A/2C and 5-HT_1A receptor agonists respectively, regardless of substrain. LLFR was predominantly A afferent fiber-mediated consistent with prominent 5-HT_2C receptor expression in the A 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 3-5 times/hour indicating an activity independent induction. Both the NMDA and AMPA/kainate receptor-mediated components of the reflex could be facilitated, and facilitation was dependent upon 5-HT receptor activation alone, not upon coincident reflex activation in the presence of 5-HT. Selective blockade of GABA_A and/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 bi-directional changes in reflex strength in a manner that is non-associative, and independent of evoked activity or activation of ionotropic excitatory and inhibitory receptors.