Activity-dependent, polyneuronal synapse elimination (ADPSE) is a programmed, regressive event in the development of the nervous system and readily studied at the neuromuscular junction, where it is complete 15-20 days after birth. Local excess, or imbalanced, protease activity is one of several possible underlying mechanisms. In this regard, thrombin mediates activity-dependent synapse loss in an in vitro model of ADPSE. To test the involvement of thrombin in vivo, we locally applied the leech thrombin-specific inhibitor, hirudin. We monitored neuromuscular behavior, correlated with acetylcholinesterase and silver nitrate histochemistry at endplates, for changes in the timecourse of in vivo synapse elimination and assayed both thrombin activity and prothrombin expression in developing muscle. Hirudin retarded elimination, without altering motor performance, uniquely at Postnatal Day 5 (P5) and maximally at P9. Reverse transcription-polymerase chain reaction (PCR) showed that neonatal muscle was a source of local prothrombin, with peak expression during the first week after birth. A specific chromogenic assay revealed that local thrombin, activated from muscle-derived prothrombin, peaked during maximal synapse remodeling.