The E. coli yeiK gene product is homologous to members of the nucleoside hydrolase family of enzymes, the physiological role of which in bacteria is still unknown. Here, the cloning, expression in milligram quantities and enzymatic characterization of YeiK as a pyrimidine-specific nucleoside hydrolase is reported. Crystals of YeiK diffract X-rays to a resolution of 1.7 A and belong to the triclinic crystal system in space group P1, with unit-cell parameters a = 44.81, b = 85.71, c = 90.68 A, alpha = 112.95, beta = 101.95, gamma = 85.92 degrees.