Inhibition of calpain activity with calpeptin or MDL-28170 prevented ITM formation. Calpain inhibition prior to training blocked the induction of LTM. Inhibiting calpain activity immediately after training impaired ITM but not LTM. Calpain activity at 6 h post-training is necessary for maintenance of 24 h LTM. Calpains function in multiple roles in associative memory formation.