The Bacillus subtilis bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) is an acidic protein of 84 amino acids that inactivates uracil-DNA glycosylase from diverse organisms (Wang, Z., and Mosbaugh, D. W. (1989) J. Biol. Chem. 264, 1163-1171). The secondary structure of Ugi has been determined by solution state multidimensional nuclear magnetic resonance. The protein adopts a single well defined structure consisting of five anti-parallel beta-strands and two alpha-helices. Six loop or turn regions were identified that contain approximately one half of the acidic amino acid residues and connect the beta-strands sequentially to one another. The secondary structure suggests which regions of Ugi may be involved in interactions with uracil-DNA glycosylase.