Generation of spontaneous rhythmic activity is a distinct feature of developing spinal networks. We report that rat embryo organotypic spinal cultures contain the basic circuits responsible for pattern generation. In this preparation rhythmic activity can be recorded from ventral interneurons and is developmentally regulated. When chronically grown in the presence of an AMPA/kainate receptor blocker, this circuit expresses long-term plasticity consisting largely of increased frequency of fast synaptic activity and reduction in slow GABAergic events. We examined whether, once this form of homeostatic plasticity is established, the network could still exhibit rhythmicity with properties similar to controls. Control or chronically treated ventral interneurons spontaneously generated (with similar probability) irregular, network-driven bursts over a background of ongoing synaptic activity. In control cultures increasing network excitability by strychnine plus bicuculline, or by raising [K+]o, induced rapid-onset, regular rhythmic bursts. In treated cultures the same pharmacological block of Cl–-mediated transmission or high-K+ application also induced regular patterned activity, although significantly faster and, in the case of high K+, characterized by slow onset due to postsynaptic current summation. Enhancing GABAergic transmission by pentobarbital surprisingly accelerated the high-K+ rhythm of control cells (though depressing background activity), whereas it slowed it down in chronically treated cells. This contrasting effect of pentobarbital suggests that, to preserve bursting ability, chronic slices developed a distinct GABAergic inhibitory control on over-expressed bursting circuits. Conversely, in control slices GABAergic transmission depressed spontaneous activity but it facilitated bursting frequency. Thus, even after homeostatic rearrangement, developing mammalian spinal networks still generate rhythmic activity.