The reversed-phase (RP) chromatographic behaviour of the macrolide antibiotics clarithromycin (Clari) and roxithromycin (Roxi) was extensively studied as a function of mobile phase composition - modified with one, two or three of organic solvents - and column temperature. The results of our study provide a satisfactory thermodynamic explanation for the retention processes of these macrolides in RP-HPLC and give additional information on the selection of optimal separation conditions for the determination of Clari in human plasma using Roxi as an internal standard and vice versa.