The capacity of the globular domain of the chicken erythrocyte linker histone H5 (GH5) to self-associate in solution has been demonstrated by chemical cross-linking with dimethyl 3,3'-dithiobis-(propionimidate) (DTBP), dithiobis(succinimidyl propionate) (DSP), and 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP). Several observations suggest that the GH5-GH5 interactions that mediate self-association are specific: (a) Incubation with each of the above reagents produces a discrete and characteristic pattern of cross-linked products; (b) GH1, the related peptide from chicken erythrocyte H1, is not cross-linked under the same conditions; (c) GH5 is not cross-linked with disuccinimidyl tartrate (DST), which has a shorter cross-linking span (6.4 A) than the other reagents (12 A); and (d) analysis of cross-linking as a function of peptide concentration provides an equilibrium constant for GH5 self-association of (4.8 +/- 1.3) x 10(3) M-1. The ability of GH5 to specifically self-associate is compatible with the proposal [Thoma, F., Koller, T., & Klug, A. (1979) J. Cell Biol. 83, 403-427] that linker histone globular domains occupy an axial position within the higher order chromatin fiber; the spatial juxtaposition of the GH5 domains at this location would be expected to promote their association and exert a stabilizing effect upon higher order chromatin structure.