A procedure of continuous-wave ultrasound (US)-induced hemolysis and reseal in solution containing water soluble protein was applied to a method for encapsulating concentrated protein solutions into resealed rat erythrocyte ghosts. To find a condition yielding a higher mean corpuscular concentration of encapsulated protein (MCC), we investigated the value of MCCs for various conditions. Additions of a small amount of plasma, Ca(2+) and Mg(2+) significantly increased MCC, whereas these additives did not alter the degree of hemolysis. It was suggested that plasma protect the molecular damages by the US, and that Ca(2+) and Mg(2+) physically stabilized the lipids of the erythrocyte membrane to fuse and reseal the pore induced by US. A maximal MCC of approximately 50 mg/mL, which is 2.5 times the reported maximum amount encapsulated by the osmotic dialysis method, was obtained without a blood-washing procedure.