In the literature, the thrombin binding aptamer GGTTGGTGTGGTTGG is generally taken as a prototype of an intramolecular guanine tetraplex of DNA. Our results, however, show that this notion is not true in aqueous solutions. This conclusion is based on a dependence of the CD spectra on aptamer concentration, migration of the aptamer in polyacrylamide gels, and the Ferguson analysis of the gel migration data. The presented data document that the aptamer forms a bimolecular tetraplex. We furthermore show that only an extension of the aptamer by a sequence containing further guanines, or an elongation of loop regions, causes that its tetraplex folding is intramolecular. (c) 2006 Elsevier Inc. All rights reserved.