Human mitochondrial NAD(P)(+)-dependent malic enzyme was overexpressed in Escherichia coli and purified by anion-exchange, ATP affinity, and gel filtration chromatography. The protein was crystallized with the hanging-drop vapor diffusion method. Many different crystal forms were observed, five of which were characterized in some detail. A 2.5-Angstrom multiple-wavelength anomalous diffraction data set and a 2.1-Angstrom native data set were collected using synchrotron radiation on crystals containing seleno-methionyl residues. These crystals belong to space group B2, with a = 204.4 Angstrom, b = 107.0 Angstrom, c = 59.2 Angstrom and gamma = 101.9 degrees. Self-rotation functions demonstrated that the tetramer of this enzyme obeys 222 symmetry.