Abstract Proteases have been used to examine the topology of proteins on various membranes. We reexamined the conditions of protease treatment for rough microsomal membranes and found that proteinase K degraded the lumenal proteins in the presence of reticulocyte lysate. The lysate treated with either heat or N-ethylmaleimide no longer promoted the degradation. The reticulocyte dependent degradation was also observed with papain, trypsin, and elastase. This activity was transiently generated by treating reticulocyte lysate short-term with trypsin. We thus concluded that a membrane perturbing factor(s) must exist in reticulocyte which is transiently activated by protease treatment.