The presence of cyclodextrins (CDs) in the mobile phase alters the chromatographic equilibria and induces a secondary chemical equilibrium associated to the chromatographic separation by HPLC. In this study the influence of the presence of CDs in the mobile phase as chemically modified beta-CDs, i.e. 2,3-di-O-methyl-beta-cyclodextrin (DMbeta-CD) and 2,3,6-tri-O-methyl-beta-cyclodextrin (TMbeta-CD) on the separation of the alkaloids norharmane, harmane and harmine is described. These beta-carboline alkaloids are chemically and structurally related and their quantitation by RP-HPLC is of interest due to their biological and pharmacological properties. Two stationary phases (methyl-, C(1) and octadecyl-, C(18)) were employed, with methanol:buffered aqueous solution and ethanol:buffered aqueous solution as mobile phases. The role of tert-butyl alcohol as a mobile phase modifier and also as an inclusion complex stabiliser was also considered. The concentrations of DMbeta-CD and TMbeta-CD vary from 0 to 17 mM. The presence of increasing amounts of CDs in the mobile phase reduces the retention factor. The changes observed in the retention factor allow the determination of the alkaloid/CD apparent association constants, whose magnitude is influenced by the chemical and structural properties of the guest molecules but also by the composition of the mobile phase. Assuming a 1:1 stoichiometry for the inclusion complexes, the apparent association constants obtained were higher for norharmane and for both DMbeta-CD and TMbeta-CD. The strength of the complexes is higher for DMbeta-CD than for TMbeta-CD and this behaviour can be explained considering the steric problems associated to the permethylated-beta-CDs. Besides significant differences in the magnitude of the apparent association constants were observed for the two stationary phases employed and thus can be related to the adsorption of CDs on the stationary phase. A significant reduction in the proportion of organic solvent in the mobile phase (50%) without a decrease in the selectivity or resolution of the separation is a favourable consequence of the presence of the CDs in the mobile phase.