The characteristics and mechanisms of synchronized firing in developing networks of cultured cortical neurons were studied using multisite recording through planar electrode arrays (PEAs). With maturation of the network (from 3 to 40 d after plating), the frequency and propagation velocity of bursts increased markedly (approximately from 0.01 to 0.5 Hz and from 5 to 100 mm/sec, respectively), and the sensitivity to extracellular magnesium concentration (0–10 mM) decreased. The source of spontaneous bursts, estimated from the relative delay of onset of activity between electrodes, varied randomly with each burst. Physical separation of synchronously bursting networks into several parts using an ultraviolet laser, divided synchronous bursting into different frequencies and phases in each part. Focal stimulation through the PEA was effective at multiple sites in eliciting bursts, which propagated over the network from the site of stimulation. Stimulated bursts exhibited both an absolute refractory period and a relative refractory period, in which partially propagating bursts could be elicited. Periodic electrical stimulation (at 1 to 30 sec intervals) produced slower propagation velocities and smaller numbers of spikes per burst at shorter stimulation intervals. These results suggest that the generation and propagation of spontaneous synchronous bursts in cultured cortical neurons is governed by the level of spontaneous presynaptic firing, by the degree of connectivity of the network, and by a distributed balance between excitation and recovery processes.