Using an assay of the rate of crystal growth of calcium [14C]oxalate monohydrate, we ascertained that the factor responsible for more than 90% of the crystal growth inhibition in human urine is a nondialyzable macromolecule. We have purified this factor using DEAE-cellulose chromatography, followed by Bio-Gel P-10 column chromatography with 50% formamide as the eluent, and finally by gel permeation chromatography. About 5 mg of the inhibitor was obtained from normal 24-h adult urine, with 16% recovery of the original inhibitory activity. The inhibitor isolated was found to be a highly acidic glycoprotein with Mr = 1.4 X 10(4). It is rich in acidic amino acids, including gamma-carboxyglutamic acid, and contains few aromatic and basic amino acids. Two or three phosphate groups are covalently linked to the inhibitor. In the presence of the inhibitor, the kinetics of calcium oxalate monohydrate crystal growth inhibitor showed that the macromolecule binds to the crystal surface according to a Langmuir adsorption isotherm with a dissociation constant, Kd = 5.3 X 10(-7) M. The inhibitor readily formed an insoluble monolayer at the air-water interface and showed an unusually high surface stability with a collapse pressure of 41.5 dynes/cm. The urinary inhibitor closely resembled in all properties the calcium oxalate monohydrate crystal growth inhibitor that we had isolated from human embryonic kidney tissue culture medium (Nakagawa, Y., Margolis, H. C., Yokoyama, S., Kézdy, F. J., Kaiser, E. T., and Coe, F. L. (1981) J. Biol. Chem. 256, 3936-3944).