Cytoskeletons of erythrocytes from the toad Bufo marinus are composed of a surface-associated cytoskeleton that encapsulates the annular bundle of microtubules known as the marginal band (MB) and the centrally located nucleus. As seen by phase-contrast microscopy, the microtubules (MTs) of the MB remain tightly bundled after cell lysis without the need for added stabilizing factors. The integrity of this structure suggested that in addition to MTs other components were present in the MB and were responsible for its stability. Thin (less than 18 nm) platinum-carbon (Pt-C) replicas of freeze-dried cytoskeletons prepared by using a modified Balzers 300 system provided a novel method of sample preparation for a high-resolution study of the ultrastructure of the MB. Electron micrographs of replicas revealed that, the MTs of the MB displayed numerous filamentous projections which, when viewed in stereo, appear as side-arm connections between adjacent MTs. Immunofluorescence data show that monospecific antibodies to tubulin and to MT-associated protein 2 (MAP2) from brain each detect cross-reactive material in the MB. The combination of immunogold cytochemistry with Pt-C replication provided the increased resolution required to identify the individual structures recognized by antibodies to tubulin and MAP2. As expected, antitubulin labeled the MTs of the MB. However, anti-MAP2 antibodies were localized specifically to the cross-bridging filaments between adjacent MTs. Thus, a MAP2-like protein was identified in situ as the crossbridging filament that bundles MTs to form a stable MB.