Recognition of the unique internal assembly origin on tobacco mosaic virus (TMV) RNA by the disk aggregate of the viral coat protein probably involves an extended region of the RNA (larger than that coated by a single disk) folded into a specific conformation. A secondary structure model is proposed for the RNA preferentially coated by limiting amounts of coat protein disks on the basis of partial nuclease digestion data. Part of this sequence can form three symmetrically spaced hairpins with marginally stable base paired sequences at the tips of the stems. The pattern of progressive protection of the RNA from nuclease attack during assembly suggests that these three hairpins are successively coated by the first three disks to add. The spacing of these hairpins is identical to that of three hairpins in the pseudo assembly origin (part of the coat protein gene homologous to the assembly origin). In Ni 2519, a TMV mutant whose assembly is defective at high temperature because it can no longer discriminate between the true and pseudo assembly origins, a point mutation has occurred near the tip of the third metastably base paired stem of the true assembly origin which would disrupt its structure and alter one copy of a repeated heptanucleotide. This suggests an important role for the ordered and cooperative recognition of successive loops in determining the specificity of assembly.