The alpha-amino acids 4(S)-hydroxyproline and 4(R)-hydroxyproline have been studied under isolation conditions in gas phase using laser-ablation molecular-beam Fourier transform microwave spectroscopy. Two conformers of each molecule have been detected in the jet-cooled rotational spectrum. The most stable conformer in both molecules exhibits an intramolecular N...H-O hydrogen bond (configuration 1) between the hydrogen atom of the carboxylic group and the nitrogen atom. The second conformer is characterized by an intramolecular N-H...O=C hydrogen bond (configuration 2). The conformers of 4(R)-hydroxyproline adopt a C(gamma)-exo puckering, while those of 4(S)-hydroxyproline present a C(gamma)-endo ring conformation. These ring conformations, which show the same propensity observed in collagen-like peptides, are stabilized by additional intramolecular hydrogen bonds involving the 4-hydroxyl group, with the exception of the most stable form of 4(S)-hydroxyproline for which a n-pi interaction between the oxygen atom of the 4-hydroxyl group and the carboxyl group carbon seems to be established. A gauche effect could be also contributing to stabilize the observed conformers.