The practical and theoretical aspects of three-dimensional homonuclear HartmannHahn-nuclear Overhauser enhancement spectroscopy (31) HOHAHA-NOESY) are presented and illustrated using the protein α1-purothionin as an example. A number of sequences are proposed with frequency selection either in the F1 dimension only or in both the F1 and the F2 dimensions, and their relative merits are discussed, particularly with respect to spectral resolution and measurement time. In addition, the nature of the various signals arising in 3D homonuclear spectroscopy, methods of evaluation of 3D HOHAHA-NOESY spectra, and the expected patterns of 3D cross peaks for different secondary structure elements in proteins are considered.