For a short time during development immature circuits in the spinal cord and other parts of the central nervous system spontaneously generate synchronous patterns of rhythmic activity. In the case of the spinal cord, it is still unclear how strongly synchronized bursts generated by interneurones are associated with motoneurone firing and whether the progressive decline in spontaneous bursting during circuit maturation proceeds in parallel for motoneurone and interneurone networks. We used organotypic cocultures of spinal cord and skeletal muscle in order to investigate the ontogenic evolution of endogenous spinal network activity associated with the generation of coordinate muscle fibre contractions. A combination of multiunit electrophysiological recordings, videomicroscopy and optical flow computation allowed us to measure the correlation between interneurone firing and motoneurone outputs after 1, 2 and 3 weeks of in vitro development. We found that, in spinal organotypic slices, there is a developmental switch of spontaneous activity from stable bursting to random patterns after the first week in culture. Conversely, bursting recorded in the presence of strychnine and bicuculline became increasingly regular with time in vitro. The time course of spontaneous activity maturation in organotypic slices is similar to that previously reported for the spinal cord developing in utero. We also demonstrated that spontaneous bursts of interneurone action potentials strongly correlate with muscular contractions only during the first week in vitro and that this is due to the activation of motoneurones via AMPA-type glutamate receptors. These results indicate the occurrence in vitro of motor network development regulating bursting inputs from interneurones to motoneurones.