The chromatographic performance of a monolithic column (Chromolith RP-18e) was comprehensively examined in the isocratic separation of ten beta-blockers, using ACN-water mobile phases, and compared with the performance of three microparticulate RP columns manufactured with different types of silica: Spherisorb ODS-2, Kromasil C18 and XTerra MS C18. The comparison considered the analysis time, selectivity, peak shape (column efficiency and asymmetry) and resolution, and was extended to a wide range of mobile phase compositions. The Chromolith column showed good performance for the analysis of beta-blockers with regard to the packed columns. In terms of selectivity and analysis time, the greatest similarity was found between the Chromolith and XTerra columns. The addition of a silanol blocking agent (0.1% triethylamine) to both Chromolith and Spherisorb columns yielded, apparently, a similar blocking degree of the silanol groups (based on the similar peak shapes), and gave rise to similar selectivity.