The ability of independent pyr.pur tracts to participate in triplex formation has been investigated in linear plasmids. The pyr.pur tract could be positioned at the ends of the plasmids or internally by a suitable choice of restriction enzyme. Dimer formation between plasmids was monitored by mobility shifts on agarose gels as well as by direct visualization in the electron microscope. Linear dimers and X and Y structures were observed. Control experiments showed that a pyr.pur tract was essential and was consistent with triplex formation in which the two pyrimidine strands were antiparallel. These structures were formed at pHs between 4 and 6, but once formed they remained stable up to pH 7. Spermine was required for formation of dimers at low ionic strength, but once formed the dimers remained stable in the absence of spermine. Additional linear plasmids were constructed with pyr.pur tracts at both ends; these formed structures at pH 4 which had mobilities identical to those of open circles. Triplex formation of this type may serve as a good model for loop formation in eukaryotic chromosomes.