Organotypic cerebellar cultures derived from newborn mice were simultaneously exposed to activity-blocking agents and neurotrophins for 2 weeks. Activity-blocked explants treated with the TrkB receptor ligands BDNF and neurotrophin-4 (NT-4) developed a full complement of Purkinje cell inhibitory axosomatic synapses, as defined ultrastructurally, and displayed control spontaneous cortical discharge rates after recovery from activity blockade. Otherwise untreated activity-blocked cultures and activity-blocked cultures exposed to the TrkC receptor ligand NT-3 had reduced inhibitory synapse development and persistent cortical hyperactivity after recovery. The added TrkB receptor ligands did not induce axonal sprouting to account for increased inhibitory synaptogenesis. Addition of neurotrophins to untreated cerebellar cultures did not increase the complement of Purkinje cell axosomatic synapses. Exposure of cerebellar cultures to a combination of antibodies to BDNF and NT-4 resulted in reduced inhibitory synapse formation, similar to the effects of activity blockade, indicating the necessity for endogenous neurotrophins for development of the full complement of inhibitory synapses in the presence of neuronal activity. Application of antibodies to BDNF and NT-4 to cerebellar explants exposed to picrotoxin to increase neuronal activity prevented the hyperinnervation of Purkinje cell somata by inhibitory terminals characteristic of cultures exposed to picrotoxin alone. These results are consistent with the concept that TrkB receptor ligands promote inhibitory synaptogenesis. The ability of neurotrophins to substitute for neuronal activity in encouraging development of inhibitory synapses may have therapeutic implications.