Low dose electron diffraction and imaging techniques have been applied to the study of the crystalline structure of gp32*I, a DNA helix destabilizing protein derived from bacteriophage T4 gene 32 protein. A quantitative analysis of intensities from electron diffraction patterns from tilted, multilayered gp32*I crystal has provided the unit cell thickness of the crystal. The three-dimensional phases indicate that the space group P2(1)2(1)2. By taking into account the unit cell volume and the solvent content in the crystal, it was deduced that there is one gp32*I molecule in each asymmetric unit. A projected density map of unstained, glucose-embedded gp32*I crystal was synthesized with amplitudes from electron diffraction intensities and phases from electron images with reflections out to 7.6 A. Because of the similarity in the scattering density between glucose and protein, this projected map cannot be interpreted with certainty. A low resolution three-dimensional reconstruction shows that the protein molecule is about 90 A long and about 20 A in diameter. Because the dimer is formed around a dyad axis, the protein molecules comprising it must be arranged head-to-head. This dimeric arrangement of the proteins in the unit cell may be implicated as one of the conformational states of this protein in solution.