Regularities of ion-pair HPLC of oligonucleotides were studied. An algorithm was suggested for the calculation of the acetonitrile gradient providing proportionality between the number of nucleotide residues and the retention time of the oligonucleotide (tR) (equidistant separation of oligonucleotides). The algorithm is based on extrapolation of the results of separation of a test mixture of five oligonucleotides varying in length from 10 to 30 units. It was shown that the logarithmic gradient of acetonitrile C0 + m ln(t) leads to the equidistant separation of oligonucleotides. The separation is characterized by two measured parameters: the retention time step and the extent of equidistantness. A functional dependence of the measured parameters on two varying parameters of the logarithmic function (C0 and m) was established experimentally. A method for calculating the expected retention time of an oligonucleotide from its nucleoside composition was suggested.