The structure-retention correlation of various C24 bile acid isomers was studied by the addition of methyl beta-cyclodextrin (Me-beta-CD) to mobile phases in reversed-phase high-performance liquid chromatography (HPLC). The compounds examined include a series of monosubstituted bile acids related to cholanoic acids differing from one another in the position and configuration of an oxygen-containing function (hydroxyl or oxo group) at the position C-3, C-6, C-7, or C-12 and the stereochemistry of the A/B-ring fusion (trans 5 alpha-H and cis 5 beta-H) in the steroid nucleus. The inclusion HPLC with Me-beta-CD was also applied to biologically important 4 beta- and 6-hydroxylated bile acids substituted by three to four hydroxyl groups in the 5 beta-steroid nucleus. These bile acid samples were converted into their fluorescence prelabeled 24-pyrenacyl ester derivatives and chromatographed on a Capcell Pak C18 column eluted with methanol-water mixtures in the presence or absence of 5 mM Me-beta-CD. The effects of Me-beta-CD on the retentions of each compound were correlated quantitatively to the decreasing rate of capacity factors and the relative strength of host-guest interactions. On the basis of the retention data, specific and nonspecific hydrogen-bonding interactions between the bile acids and the Me-beta-CD were discussed.