Based on the recent finding that a peroxidase-conjugated calsequestrin probe retains its biological binding properties, we have used it in two-dimensional blot and cell overlay procedures. Labeling of nitrocellulose replicas of electrophoretically separated microsomal proteins from predominantly fast versus slow fibres revealed self-aggregation of fast calsequestrin with molecular species of differing isolectric points. Incubation of transverse cryosections showed restricted calsequestrin interactions with elements in the fibre interior, mostly representing calsequestrin itself. This confirms that fast calsequestrin exists in large aggregates under native conditions and demonstrates the usefulness of blot and cell overlay techniques in identifying and locating supramolecular protein assemblies.