AbstractA means of rapidly renaturing denatured protein was devised and evaluated. Three liquids were laminarly layered in a centrifuge tube, in which two solutions sandwiched liquid paraffin so as to form a pseudolipid bilayer. Denatured and aggregted protein placed on the upper surface of liquid paraffin was renatured as it passed through liquid‐paraffin layer into the renaturation buffer during the centrifugation. The aggregated and denatured protein selectively passed through the liquid‐paraffin layer, whereas other solutions, such as chaotropic agents or organic solvent, could not. This means that a rapid dilution condition favorable for protein renaturation was realized in a small scale. Aggregated and denatured BSA and ribonuclease A were renatured and resolubilized as they passed through the liquid‐paraffin layer into an appropriate renaturation buffer solution. This method was also applied to the rapid heme reconstitution of myoglobin from Feprotoporphyrin IX to Zn‐protoporphyrin IX. © 1994 John Wiley&S