The protease inhibitor leupeptin prevented multiplication of the human coronavirus strain 229E in cultures of MRC-C cells. The IC50 of leupeptin in plaque reduction tests was 0.4 micrograms/ml, whereas growth of host cells was unaffected by leupeptin at 50 micrograms/ml. Inhibition of plaque formation could be prevented by the addition of proteases to the overlay medium. In single-cycle growth experiments, leupeptin reduced virus yield only if added within 2 h of infection, indicating its action on an early stage of virus replication.