Single crystals of the protein lysin (Mr = 16,070) from the spermatozoa of the red abalone (Haliotis rufescens) have been obtained by the vapor diffusion technique, using as precipitants a 32.5% saturated solution of (NH4)2SO4 (incubation at 18 degrees C) or a 5% w/v polyethyleneglycol 8000 solution (incubation at 29 degrees C), both in Bis-Tris-iminodiacetic acid buffers of pH 7.0. The addition to the droplets of EDTA, other carboxylate-containing polyanions, and/or organic solvents improved the size and quality of the crystals and, especially with (NH4)2SO4, addition of EDTA, and/or organic solvents produced a change in crystal habit which resulted in crystals more elongated in the b direction. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with a = 52.3 A, b = 46.0 A, and c = 81.5 A and one molecule per asymmetric unit. The crystals diffract to 2.3 A resolution. The molecular structure of lysin is relevant to the nonenzymatic mechanism by which the protein dissolves a hole in the egg vitelline layer during fertilization.