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Three unusually large myofibrillar proteins (Mr, > 500,000) are present in major amounts in the striated muscle of various species. the molecular properties and localization of two of these proteins (Mr, approximately 1 X 10(6)), named "titin," have been reported recently [Wang, K., McClure, J. & Tu, A.(1979) Proc. Natl. Acad. Sci. USA 76, 3698-3702]. We have now purified the third protein (Mr, approximately 5-6 X 10(5)), designated "band 3," from rabbit psoas myofibrils. Band 3 is distinct chemically and immunochemically from titin and myosin heavy chain. Immunofluorescent localization studies of band 3 in glycerinated rabbit psoas myofibrils indicated a biphasic distribution. In sarcomeres 1.8-3.7 micrometer long, band 3 is localized predominately on the N2 line in the phase-contrast lucent region of the I band. Quantitative analysis of light micrographs confirmed the unique behavior of N2 lines described in early …