The organization of telomeric chromatin differs from that of bulk chromatin in some peculiar features, such as the unusually short nucleosomal spacing found in vertebrates. Telomeric DNAs are straight, since they consist mostly of 6-8-bp repeated sequences, therefore out of phase with the B DNA period. This feature should be of relevance in nucleosome formation, suggesting the usefulness of studying simple model systems of nucleosome assembly. We reconstituted nucleosomes in vitro, by using purified histone octamers and/or by octamer transfer from chicken erythrocyte nucleosomes, onto telomeric sequences from human, Arabidopsis thaliana, and Saccharomyces cerevisiae. All of these telomeres contain GGG and GGT triplets but are characterized by different repeat lengths (6, 7, and 8 bp). The free energies involved in the association process are the highest among the biological sequences so far assayed, suggesting a main role of DNA flexibility in the assembly of telomeric chromatin. Digestion studies with DNase I, hydroxyl radicals, exonuclease III, and lambda exonuclease indicate that telomeric nucleosomes are characterized by multiple translational positioning without rotational phasing, whereas the telomeric DNA folding around the histone octamer shows the canonical periodicity of about 10.2 bp. The experimental results and a theoretical simulation of DNase I digestion indicate a multiple nucleosome positioning with the periodicity of telomeric DNA. This suggests a main role of local chemical recognition between telomeric sequences and the histone octamer in nucleosome assembly.