A study of the regular secondary structure elements of recombinant human interleukin-1 beta has been carried out using NMR spectroscopy. Using a randomly 15N labeled sample, a number of heteronuclear three- and two-dimensional NMR experiments have been performed, which have enabled a complete analysis of short-, medium-, and long-range NOEs between protons of the polypeptide backbone, based on the sequence-specific resonance assignments that have been reported previously [Driscoll, P. C., Clore, G. M., Marion, D., Wingfield, P. T., & Gronenborn, A. M. (1990) Biochemistry 29, 3542-3556]. In addition, accurate measurements of a large number of 3JHN alpha coupling constants have been carried out by two-dimensional heteronuclear multiple-quantum-coherence-J spectroscopy. Amide NH solvent exchange rates have been measured by following the time dependence of the 15N-1H correlation spectrum of interleukin-1 beta on dissolving the protein in D2O solution. Analysis of these data indicate that the structure of interleukin-1 beta consists of 12 extended beta-strands aligned in a single extended network of antiparallel beta-sheet structure that in part folds into a skewed six-stranded beta-barrel. In the overall structure the beta-strands are connected by tight turns, short loops, and long loops in a manner that displays approximate pseudo-three-fold symmetry. The secondary structure analysis is discussed in the light of the unrefined X-ray structure of interleukin-1 beta at 3-A resolution [Priestle, J. P., Schär, H.-P., & Grütter, M. G. (1988) EMBO J. 7, 339-343], as well as biological activity data. Discernible differences between the two studies are highlighted. Finally, we have discovered conformational heterogeneity in the structure of interleukin-1 beta, which is characterized by an exchange rate that is slow on the NMR chemical shift time scale.