The conformation of hydroxy-L-proline in D2O solution has been studied by analysing its 1H chemical shifts and spin-lattice relaxation times perturbed by the binding of lanthanide(III) ion. Spin couplings, including a large long-range coupling constant (2.22 Hz) between the protons bonded to the β- and δ-carbons, remain unaltered by the binding of LaIII. Thus, conformational changes upon complex formation are absent. The assignment of protons in this study which utilizes the lanthanide probe is in agreement with previous result from analysis of the coupling constants. The conformations estimated independently by these two approaches are similar, although from a more quantitative point of view the puckering angles ω1, (between the planes formed by Cα, Cβ, and N and by Cβ, Cδ, and N) and ω2(between the planes formed by Cβ, Cδ, and N and by Cβ, Cγ, and Cδ) differ by ca. 20 and 15°, respectively, between the two conformations.