The affinity of estradiol derivatives for the estrogen receptor (ER) depends strongly on nature and stereochemistry of substituents in C(11) position of the 17beta-estradiol (I). In this work, the stereochemistry effects of the 11alpha-OH-17beta-estradiol (III(alpha)) and 11beta-OH-17beta-estradiol (III(beta)) were investigated using CID experiments and gas-phase acidity (DeltaH degrees (acid)) determination. The CID experiments showed that the steroids decompose via different pathways involving competitive dissociations with rate constants depending upon the alpha/beta C(11) stereochemistry. It was shown that the fragmentations of both deprotonated [III(alpha)-H]- and [III(beta)-H]- epimers were initiated by the deprotonation of the most acidic site, i.e. the phenolic hydroxyl at C(3). This view was confirmed by H/D exchange and double resonance experiments. Furthermore, the DeltaH degrees (acid) of both epimers (III(alpha) and III(beta)), 17beta-estradiol (I), and 17-desoxyestradiol (II) was determined using the extended Cooks' kinetic method. The resulting values allowed us to classify steroids as a function of their gas-phase acidity as follows: (III(beta)) >> (II) > (I) > (III(alpha)). Interestingly, the alpha/beta C(11) stereochemistry appeared to influence strongly the gas-phase acidity. This phenomenon could be explained through stereospecific proton interaction with pi-orbital cloud of A ring, which was confirmed by theoretical calculation.