The multiwavelength anomalous diffraction (MAD) method for phase evaluation is now widely used in macromolecular crystallography. Successful MAD structure determinations have been carried out at the K or L absorption edges of a variety of elements. In this study, we investigate the anomalous scattering properties of uranium at its MIV (3.326 Å) and MV (3.490 Å) edge. Fluorescence spectra showed remarkably strong anomalous scattering at these edges (f ؍ ؊70e, f ؍ 80e at the MIV edge and f ؍ ؊90e, f ؍ 105e at the M V edge), many times higher than from any anomalous scatterers used previously for MAD phasing. However, the large scattering angles and high absorption at the low energies of these edges present some difficulties not found in typical crystallographic studies. We conducted test experiments at the MIV edge with crystals of porcine elastase derivatized with uranyl nitrate. A four-wavelength MAD data set complete to 3.2-Å Bragg spacings was collected from a single small frozen crystal. Analysis of the data yielded satisfactory phase information (average difference of 0 T ؊ 0 A for replicated determinations is 32°) and produced an interpretable electron-density map. Our results demonstrate that it is practical to measure macromolecular diffraction data at these edges with current instrumentation and that phase information of good accuracy can be extracted from such experiments. We show that such experiments have potential for the phasing of very large macromolecular assemblages.MAD ͉ protein crystallography ͉ resonance scattering ͉ x-ray absorption X -ray absorption increases sharply as the energy of incident x-rays meets the transition energy needed to excite an electron from an atomic orbital into the continuum. A prominent peak of absorption, known as a ''white line,'' can also appear at an absorption edge. These features enhance the intrinsic atomic absorption to produce a very rapid change with respect to wavelength in the vicinity of the absorption edge. White line features result from the stimulated resonant transition of an electron from a bound atomic state to an unoccupied molecular orbital. A resonant modulation of x-ray scattering accompanies the dispersion of x-ray absorption; these anomalous scattering increments to the normal scattering include an imaginary part, fЈЈ, that is proportional to the x-ray absorption spectrum and a real part, fЈ, that is related to fЈЈ by the Kramers-Kronig dispersion integral. The method of multiwavelength anomalous diffraction (MAD) takes advantage of such anomalous dispersion of x-ray scattering to accomplish de novo phasing of macromolecular crystal structures (1). In a MAD experiment, diffraction data are measured at several different wavelengths chosen in the vicinity of the absorption edge of a certain anomalous scatterer present in the crystal. This approach contrasts with the more conventional multiple isomorphous replacement (MIR) method, in which data sets are collected at the same single wavelength from native and different heavy-atom derivative...