Heteroduplex DNA molecules were generated in which one of the two strands contained a 7-base-pair (double-stranded) stem and 3-base-pair (single-stranded) loop. The heteroduplexes and their corresponding homoduplex parental molecules, each of approximately 260 base pairs, were used for nucleosomes reconstitution. Protection from restriction endonuclease digestion was used to probe the structure of the resulting dinucleosomes. Although 50% or more of the potential cleavage sites in the homoduplex DNAs and in the linear portion of the heteroduplex DNAs were inaccessible to nuclease digestion, no protection of the stem-loop structures was observed. The results imply that a stem-loop structure preferentially occupies the spacer region between nucleosomes, but if it is part of a core particle the stem-loop is always pointed "outward" in such a way as to be accessible to nuclease digestion.