A comparative study of syn vs anti carboxylic acids in hybrid peptides based on experimental electron density studies and theoretical calculations shows that, in the anti form, all three bond angles surrounding Ccarboxyl of the -COOH group are close to ∼120°, as expected for a C-sp2 atom, whereas in the syn form, the ∠Cα-C(O)-Ohydroxyl angle is significantly smaller by 5-10°. The oxygen atom in the carboxyl group is more electronegative in the anti form, so the polarity of the acidic O-H bond is higher in the anti form compared to the syn form, as observed within the limitations of H atom treatment in X-ray diffraction. Consequently, the investigated anti carboxylic acid forms the strongest O-H···O hydrogen bond among all model compounds. Furthermore, according to natural bond orbital analysis, the oxygen lone pairs are clearly nonequivalent, as opposed to the general notion of hybridization of equivalent sp2 and sp3 lone pairs on carbonyl or hydroxyl oxygen atoms. The hybridization of the lone pairs is directly related to the directionality and strength of hydrogen bonds.