Rabbit skeletal muscle tropomyosin can be crosslinked in the native state by the use of 5,5'-dithiobis(2-nitrobenzoate), which forms disulfide bonds between the two subunits. Using polyacrylamide gel electrophoresis in sodium dodecyl sulfate we have shown that this crosslinking goes to completion over a wide range of protein concentration, ionic strength, and reagent concentration. Crosslinks are not formed in denaturing solvents such as sodium dodecyl sulfate and guanidine hydrochloride despite the fact that the same number of SH groups react as in the native state (2.3 +/- 0.2). The sedimentation coefficients of crosslinked and non-crosslinked samples are identical. Thus, crosslinks are formed between corresponding cysteines on different chains of the same molecule. This provides strong evidence for a model of chain interaction with both chains in register. Evidence has also been obtained that rabbit skeletal tropomyosin is composed only of alphaalpha and alphabeta subunits rather than a random mixture of chains.