Abstract The dinucleotides ApGp, CpGp, and UpGp and seven trinucleotides, ApApGp, ApCpGp, ApUpGp, CpApGp, Cp-CpGp, UpApGp, and UpUpGp, were obtained by Taka-Diastase ribonuclease T1 digestion of yeast RNA and subsequent chromatographic separation with DEAE-Sephadex followed by Dowex 1-X2. The present chromatographic method is suitable for large scale preparation of pure di- and trinucleotides. The absorption spectra and hypochromicities of these oligonucleotides were measured at pH 1, 7, and 13. It is found that the order of the stacking effect in the dinucleotides is ApGp g CpGp g UpGp, which is different from a conclusion drawn from hypochromicity and optical rotatory dispersion data recently reported by Warshaw and Tinoco for the corresponding dinucleoside phosphates. This difference suggested that the additional 3'-terminal phosphate residue has an appreciable effect on the conformations of the dinucleoside phosphates. For the trinucleotides, the hypochromic effect is found to decrease in the order, ApApGp g ApCpGp ≃ CpApGp g UpApGp g CpCpGp g ApUpGp g UpUpGp. The optical rotatory dispersion spectra of two pairs of isomers of known sequence are presented to show the dependence of optical rotatory dispersion on sequence in these oligonucleotides. The possibility of synthesis of polyribonucleotides containing definite repeating di- or trinucleotide sequences is also considered as a potentially important consequence of the present success in large scale preparation of these oligonucleotides.