Organotypic cerebellar cultures derived from newborn mice were continuously exposed to medium containing tetrodotoxin and elevated levels of magnesium to block all electrical activity. After 2 weeks in vitro, no activity was evident during the first 15–20 minutes following transfer to a recording medium without blocking agents. Thereafter, cortical discharge rates increased until a state of sustained hyperactivity was reached. Ultrastructural examination of such cultures revealed a reduction of inhibitory Purkinje cell somatic synapses to half the control value along with an even greater reduction of axodendritic synapses (largely inhibitory) in the cortical neuropil. No loss of axospinous synapses (excitatory) was evident. These results support the concept that spontaneous neuronal activity is necessary for the full development of inhibitory circuitry. © 1994 Wiley-Liss, Inc.