The effect of temperature on the retention and multiple separation of hydrocortisone, testosterone, 17 alpha-methyltestosterone, prednisone, cortisone and 17 alpha-hydroxyprogesterone in reversed-phase liquid chromatography has been studied. Capacity factors (k') of the steroids were measured using a mobile phase modified with different concentrations of beta-cyclodextrin (from 0-16 mM), a fixed solvent composition (acetonitrile-water) and a wide range of column temperatures (from 5-80 degrees C). The plots of capacity factors vs. reciprocal of absolute temperature are nonlinear in every case when mobile phase modified with beta-cyclodextrin was used. Particularly strong nonlinearity was observed at lower temperature and at higher beta-cyclodextrin concentration. The complex chromatograms were evaluated using optimization parameters such as capacity factor of the last-eluted peak (k'max), the smallest resolution between adjacent peaks (Rs; min) and relative resolution product (r). The results presented describe precisely the role of temperature in high performance liquid chromatography systems in which mobile phases modified with cyclodextrin were used.