Fodrin is an axonally transported F-actin crosslinking rod-shaped multidomain protein having a length of 200 nm. It is though to be related to erythroid spectrin (220,000 and 240,000 Mr) as it is built as a tetramer derived from a heterodimer comprised of two large subunits (235,000 and 240,000 Mr). A panel of 24 monoclonal antibodies has been used to probe the fodrin structure by direct microscopical decoration and identification of large tryptic fragments retaining antigenicity. The combined results unambiguously define the fodrin organization and show it to be strongly related to erythrocyte spectrin. They demonstrate the heterodimer organization, the head-to-head association of dimers, the protease-resistant domain structure of each subunit and indicate that different termini of the polypeptides are localized at the free F-actin binding ends and the heterodimer association sites. They also show that within the same mammalian species even the highly related 240,000 Mr subunits of fodrin and spectrin are immunologically distinct, most likely reflecting different gene products.