The peptide fragment Arg-Lys-Glu-Val-Tyr (SP5) is the active site fragment of splenin (formerly thymopoietin III), an immunoregulatory polypeptide isolated from bovine spleen. Like its parent polypeptide, SP5 also induces the differentiation of T and B lymphocytes. We report here an NMR investigation of the conformational properties of this active fragment in aqueous solution. All the observed NH and CH resonances of SP5 have been assigned by one-dimensional and two-dimensional NMR techniques. The variation of chemical shifts with pH, the individual amide hydrogen exchange rates, and the vicinal NH-C'H coupling constants have been measured. The data are compatible with the assumption of a highly motile dynamic equilibrium among different conformations, some of which are stabilized by internal hydrogen bonding involving the participation of G1u3-NH, Val4-NH, and Tyr5-NH in the backbone and of the guanidino N'H proton of the Arg1 side chain. These observations provide an insight into the conformational tendencies of SP5 in aqueous solutions. © 1983, American Chemical Society. All rights reserved.