SUMMARY A quick‐freeze, freeze‐substitution method is described which employs glutaraldehyde as well as osmium tetroxide (OsO4) in a ‘double‐fixation’ protocol comparable to that used for conventional transmission electron microscopy. Cultured cells are quick‐frozen in Freon 22 and freeze‐substituted in an ethanolic solution of glutaraldehyde. Specimens destined for TEM are postfixed in OsO4 in acetone, embedded in Epon‐Araldite, and sectioned. This method yielded ultrastructural preservation which was comparable to that obtained from methods employing OsO4 alone as a freeze‐substitution fixative. However, if glutaraldehyde is used alone as a freeze‐substitution fixative, specimens can be processed for immunocytochemistry without additional treatment with permeabilizing agents.