In recent years the structures of many small proteins have been determined by twodimensional NMR, using methods pioneered by Wiithrich et al. (I ). These methods depend exclusively on data from proton NMR experiments and despite their success it has become clear that they are more difficult to apply and less likely to work for proteins with molecular weights of more than about 15,000. The deleterious effects of greater molecular size arise both from the large number of signals and from the increase in the linr~tidths of the signals: among other things these effects result in spectra which have a much higher degree of overlap. This is particularly acute in proteins with a high helical content, in which, for example, the majority of the amide protons are expected to resonate within a range of just one part per million. Several different strategies for overcoming this problem have been proposed. Among the most promising are those that depend upon nonspecific near-complete isotopic substitution of 15N for 14N or 13C for “C, in conjunction with two-dimensional NMR experiments which exploit the shift range of the heteronuclei to separate the proton signals (2-8). An alternative and potentially very powerful strategy is the use of threedimensional NMR to alleviate spectral overlap (9-12). The combination of threedimensional NMR with heteronuclear labeling is proving particularly effective (1318), and an important example ofthis approach is the 3D NOESY-HMQC experiment, developed by Marion et ~11. (14) and Zuiderweg and Fesik (15) for use with “Nlabeled proteins. This experiment may be thought of as generating a 15N-edited NOESY spectrum, with signals spread out in a third dimension according to the lSN shift of the directly coupled amide nitrogens. More formally, a proton H, which has an NOE to an amide proton Hi, will generate a cross peak in the NOESY-HMQC experiment centered at frequency coordinates (h,, nb, hi,), where h, and hb are the chemical shifts of H, and Hh, respectively, and nb is the chemical shift of the “N nucleus that is directly coupled to Hi,. It is clear that by spreading the signals according to the nitrogen shifts this experiment would allow the Ha-HI, interaction to be identified even if there were one or more additional protons which had the same shift as Hi,. However, if H, and Hi, themselves overlap the NOESY-HMQC experiment does not allow an NOE interaction between them to be detected: in this case the presence of the characteristic cross peak at (h,, nb, hb) would be masked by the coincident and more intense “di-