Peptidyl-tRNA hydrolase from Escherichia coli, a monomer of 21 kDa, was overexpressed from its cloned gene pth and crystallized by using polyethylene glycol as precipitant. The crystals are orthorhombic and have unit cell parameters a = 47.24 A, b = 63.59 A, and c = 62.57 A. They belong to space group P2(1)2(1)2(1) and diffract to better than 1.2 A resolution. The structure is being solved by multiple isomorphous replacement.