The underivatized enantiomers of carbuterol, chemically known as [5-[2-[(1, 1-dimethylethyl)amino]-1-hydroxyethyl]-2hydroxyphenyl]urea, an isoprenaline like oral β2-adrenoceptor agonist widely used as bronchodilator, have been separated in chiral RP-HPLC isocratic system employing a conventional octadecylsilica packed column and chiral, helically self-assembled, neutral, coordinatively unsaturated chelate(-)(M)(λ, Λ)-[4,4′-(1S)-methyl-(2R)-propylethane-diyldiimino)bis(pent-3-en-2-onato)nickel(II) as the chiral selector in the mobile phase comprising acetonitrile-sodium acetate solution. The changes in observed enantioselectivity and resolution of carbuterol stereoisomers caused by organic modifier concentration or chiral selector adsorption were investigated. The formation of hydrogen bonded supramolecules by involving three molecules of the helically self-assembled chiral selector and a single molecule of individual enantiomer of carbuterol have been suggested. Hybridized orientation of interacting molecules in the supramolecular selectand-selector system was exhibited on results of extended analyses of developed chiral recognition model. Considering collected chromatographic and molecular modelling data it has been postulated that process of supramolecular association of the carbuterol enantiomers prevailing in the volume phase of employed RP-HPLC mode. An inverted elution order of(R-)and(S-)enantiomers of analyte have been registered in the presented studies, in contrast to previously reported retention of other sympathomimetic phenylethanolamines (e.g.noradrenaline) in the proposed chiral RP-HPLC system. The presence of a planar ureidoyl moiety in the phenylene ring of carbuterol molecule significantly enhances the stability of supramolecules formed with chiral selectors leading to improved separation of both enantiomers of this drug.