The role of histone H1 in nucleosome heterogeneity and structure has been studied using a reconstitution procedure. Histone H1 and non-histone proteins are removed selectively from enzymatically fragmented chromatin by Dowex 50W-X2 treatment. The resulting "stripped" chromatin then is reassociated with purified histone H1 using step gradient dialysis. Material reconstituted in this manner was examined by gel electrophoresis, protein cross-linking, and chromatin fingerprinting. The results demonstrate that the histone H1 molecule efficiently binds to nucleosomes with fidelity in an apparent noncooperative manner. Polynucleosomes possess two specific binding sites for histone H1 per histone octamer; the first binding site is of higher affinity than the second. The 160-base pair nuclease digestion barrier and nucleosome electrophoretic class (MIII)n are established upon binding the 1st histone H1 molecule. Upon binding the 2nd histone H1 molecule, polynucleosomes assume a highly compact conformation. The experimental approach introduced here should permit determining whether nucleosomes possess independent specific binding sites for other chromosomal proteins, and should allow reconstitution of the other electrophoretic forms of nucleosomes which we have described previously.