IscS is a widely distributed cysteine desulfurase that catalyzes the pyridoxal phosphate dependent beta-elimination of sulfur from L-cysteine and plays a central role in the delivery of sulfur to a variety of metabolic pathways. Crystals of Escherichia coli IscS have been obtained by the hanging-drop vapor-diffusion method using polyethylene glycol (PEG) as a precipitant. Initial seed crystals were obtained using PEG 6000 and sodium acetate, and diffraction-quality crystals were grown using a mixture of PEG 2000 and PEG 10 000 in the presence of sodium citrate. A complete native X-ray diffraction data set was collected from a single crystal at 103 K to a resolution of 2.1 A. The crystals belong to space group P2(1)2(1)2(1) and have unit-cell parameters a = 73.7086, b = 101.9741, c = 108.617 A (alpha = beta = gamma = 90 degrees ). Analysis of the Matthews equation and self-rotation function suggest two molecules per asymmetric unit, consistent with the presence of a single dimeric molecule.