Oligodeoxycytidine phosphorothioate (PS-dCn, n = chain length), known to show virus inhibition ability by a mechanism other than the antisense one when n approximately 20, was explored for its solution structure by circular dichroism (CD) and ultraviolet (UV) absorption spectroscopy. For PS-dC4, when the strand concentration was higher than 10 microM, the respective 288-nm positive and 265-nm negative peaks appeared in the CD spectra at slightly acidic pHs and 0 degree C in the absence of salt, which is indicative of a four-stranded structure (namely, the i-motif). Strand concentration-dependent CD spectroscopy indicated that intermolecular association is responsible for this i-motif. The formation or i-motif was also characterized by UV absorption spectroscopy, in which the dissociation of this structure caused a sharp increase in the absorbance at 275 nm and a decrease at 305 nm. By plotting this change, the Tn values were estimated to be ca. 11 and 13 degrees C at 20 and 50 microM strand concentrations, respectively. Stability of the i-motif was compared between PS-dC, P-chiral diastereoisomers, and the Sp configuration produced a more stable structure than Rp. PS-dC20 was also investigated at physiological temperature, and the respective 288-nm positive and 265-nm negative peaks appeared at slightly acidic pH: it has been suggested that intermolecular folding was predominant above ca. 1 microM and that intramolecular folding dominated at low strand concentrations such as 0.05 microM. Gel-filtration chromatography and nondenaturing gel electrophoresis provided the supporting data for the four-stranded folding of PS-dC20.