We have compared rapid freezing followed by freeze‐substitution fixation with conventional aldehyde fixation as preparative methods for the electron microscopic study of organotypic cultures of neonatal rat hippocampus. Rapid freezing by contact with a copper block chilled by liquid helium was accomplished without mechanical distortion of superficial structures, and preserved structure to a depth of at least 20 μM without visible ice crystals. Freeze‐substitution fixation in acetone/osmium tetroxide, followed by en bloc staining with tannic acid and uranyl acetate, provided satisfactory staining of cytoplasm and organelles. While both preparative techniques yielded generally satisfactory results, rapid freezing provided much better preservation of astrocytic lysosomal inclusions, and afforded new views of intermediate filament substructure. Rapid freezing and freeze‐substitution fixation seemed especially well suited to the preservation of short filamentous proteins, such as those forming the membrane cytoskeleton of dendritic spines or those associated with synaptic vesicles. The combination of rapid freezing methods and organotypic culture provides an opportunity to examine cytoplasmic structure in tissue from deep regions of the brain which had previously been inaccessible to rapid freezing techniques. © 1993 Wiley‐Liss, Inc. Copyright © 1993 Wiley‐Liss, Inc.