The structure of the nucleosome core particle, the basic structural subunit of chromatin, is well known. Although nucleosomes often appear to be positioned randomly with respect to DNA sequences, in some cases they seem to occupy precisely defined positions on the DNA. The yeast plasmid TRP1ARS1 contains three precisely positioned, stable nucleosomes, I, II and III, which are flanked by nuclease-sensitive regions. Our aim in the present study was to determine whether the positions of these three nucleosomes relate to (1) protein-DNA interactions; (2) the limited space between nuclease-sensitive regions, which is just long enough to accommodate three yeast nucleosomes (that is, boundary conditions); or (3) proximity to the putative origin of replication in one of the nuclease-sensitive regions. We have tested these alternatives by analysing the positions of nucleosomes after insertion of various lengths of DNA into this region and assembly of chromatin in vivo. Our results suggest that specific protein-DNA interactions are the most likely determinants of these nucleosome positions.