The Substance P fragment Arg1-Pro2-Lys3-Pro4 (SP1-4) has been extensively investigated by means of proton nuclear magnetic resonance at 400 MHz. The combined application of different 2D techniques and a comparison of SP1-4 with its derivative SP1-4-amide allowed the complete and unambiguous assignment of the proton NMR spectrum. Conformational data obtained from the different NMR parameters are compared with theoretical calculations. The results suggest that SP1-4 exists, at the chosen experimental conditions, as a stretched molecule.