The capillary gas chromatographic (GC) behaviour of 25 bile acids with vicinal 6,7- or 3,4-glycol structure, differing from one another in the stereochemical configuration [diaxial trans, diequatorial trans and axial—equatorial (or vice versa) cis], was studied. Methylene unit (MU) values were determined for each of the bile acids as their nine classes of hydroxyl derivatives: complete methyl ester (Me)—trimethylsilyl (TMS) and Me—dimethylethylsilyl (DMES) ether and Me—acetate (Ac) derivatives, and their so-called “mixed” cyclic alkylboronate derivatives, Me—methylboronate (MB)—TMS, Me—MB—DMES, Me—MB—Ac, Me— n-butylboronate ( nBB)—TMS, Me— nBB—DMES and Me— nBB—Ac. Changes in the MU values of each bile acid due to the different derivatizations were used for the determination of the number of hydroxyl groups and the stereochemical relationships of 1,2-diol groups in the molecules. The cis-glycol type of compound, regardles of its α,α- or β,β-configuration, formed the corresponding mixed cyclic alkylboronate derivatives. On the other hand, both the diaxial and diequatorial trans-glycols yielded a GC peak corresponding to the persilylated or peracetylated derivatives, although the latter compounds were occasionally accompanied by a second peak corresponding to the mixed alkylboronate—silyl ether derivative. The retention data would be useful for analysing bile acid mixtures extracted from biological samples.