Nucleosomes were reconstituted in vitro with several singly end-labeled restriction fragments derived from a cloned somatic-type 5S RNA gene of Xenopus laevis and purified nucleosome core particles from Xenopus cultured cells or chicken erythrocytes. Nucleosome locations were determined by digestion of the reconstitutes with exonuclease III and DNase I and were the same for all fragments investigated, extending from 20 base pairs (bp) within the 5S gene to 80 bp beyond the 3' end of the gene. Both core particles and crude nuclear extracts gave equivalent results, suggesting that no factors other than the core histones are responsible for recognition of DNA sequence during reconstitution. The histone octamer and the 5S gene-specific transcription factor TFIIIA both bind to the same region and face of 5S DNA, and nucleosome reconstitution on the 5S gene excluded binding of TFIIIA. The helical repeat of somatic-type 5S DNA in solution was measured by the band shift method and was 10.5 to 10.6 bp per turn over the region of the TFIIIA-binding site. The difference in helical repeat between DNA in solution and on the surface of the nucleosome (10.0-bp spacing between DNase I cutting sites) may explain the linking number paradox.