The influence of the nature of typical organic modifiers, used in reversed-phase systems diluted with water, on retention and selectivity of chiral acetylated diamines and acetonitriles possessing pharmacological activity was investigated. Linear, semi-logarithmic relationships between the logarithm of the retention factor, k, of the enantiomer and the volume fraction, phi, of the organic modifier in the binary aqueous-organic eluent were established for chiral stationary phases. The slope of a plot of log k vs. the modifier volume fraction depends not only on the chemical structure of the chromatographed enantiomers, but also on the nature of the modifier. Moreover, the enantioselectivity of the studied racemates also depends on the nature and concentration of the modifier and is higher for a methanol mobile phase than for acetonitrile.