To study mechanisms of nucleosome positioning, small circular plasmids were constructed, assembled into chromatin in vivo in Saccharomyces cerevisiae, and their chromatin structures were analysed with respect to positions of nucleosomes and nuclease-sensitive regions. Plasmids used include insertions of the URA3 gene into the TRP1 gene of the TRP1ARS1 circular plasmid in the same (TRURAP) or opposite (TRARUP) orientation. The URA3 gene has six precisely positioned, stable nucleosomes flanked by nuclease-sensitive regions at the 5' and 3' ends of the gene. Three of these nucleosome positions do not depend on the flanking nuclease-sensitive regions, since they are formed at similar positions in a derivative plasmid (TUmidL) that contains the middle of the URA3 sequence but not the 5' and 3' ends. These positions are probably due to protein-DNA interactions. In both TRURAP and TRARUP, the positions of the nucleosomes on the TRP1 gene were, however, shifted compared with the positions on the parental TRP1ARS1 circle and TUmidL. These changes are interpreted to be due to changes in the positions of flanking nuclease-sensitive regions that might act as boundaries to position nucleosomes. Thus, two independent mechanisms for nucleosome positioning have been demonstrated in vivo. The ARS1 region contains the 3' end of the TRP1 gene and the putative origin of replication. Since in TRURAP and TRARUP the TRP1 gene is interrupted, but the ARS1 region remains nuclease sensitive, this non-nucleosomal conformation of the ARS1 region probably reflects a chromatin structure important for replication.