Nephrocalcin (NC), an acidic glycoprotein with molecular weight 14,000, is present in urine and prevents kidney stone formation. Histoimmunochemical staining shows that NC is localized in the proximal tubles in kidneys. Isolated NC from mammalian urine, revealed at least 4 isoforms of NC (we call these isoforms NC-A, NC-B, NC-C, and NC-D in the order of elution) during DEAE cellulose column chromatography with a linear gradient of NaCl elution step. Non-stone forming people excrete more NC-A and NC-B isoforms in urine; however, more NC-C and NC-D isoforms were found in stone formers' urine. When the organic matrix was extracted from surgically removed calcium oxalate kidney stones, we found greater quantities of NC-C and NC-D isoforms than those of NC-A and NC-B isoforms. Amino acid compositions and carbohydrate contents of these 4 isoforms were similar with the exception of the gamma-carboxyglutamic acid (GLA) residues. Only the NC-A and NC-B isoforms contained residues of GLA. There were more phosphate residues present in NC-C and NC-D than in NC-A and NC-B. Upon removal of phosphate residues by alkaline phosphatase, NC-C eluted at the same salt concentrations as NC-A eluted. This indicates that the backbone protein could be similar, but the NC-C isoform is modified by excess phosphate residues. Surface tension measurements using a Lauda film balance indicated that NC-A and -B were strongly amphiphilic while NC-C and -D were less amphiphilic. NC-A has an elongated shape, and occupies a smaller area per molecule; whereas NC-C is a bulky molecule. Using NC-A as a model of a "good" inhibitor and NC-C as a model of a "poor" inhibitor, both bound with 4 atoms of Ca2+ per molecule as investigated by equilibrium dialysis method, 31P-NMR, and electron spin resonance spectrometry. Isoforms A and B changed their conformation upon Ca2+ binding, but C and D did not change their conformation. All these observations suggest that isoforms A and B are strong inhibitors of calcium oxalate monohydrate (COM) crystal growth and aggregation. However, isoforms C and D act as promotors for COM crystal growth-kidney stone formation. Measuring the amount of NC in urine from renal cell carcinoma patients and from NC isolated from a supernatant of a primary renal cell carcinoma cells demonstrated the amount of NC increased with disease progression.