We have investigated urine protein inhibitors of calcium oxalate crystallization to determine whether variations in these proteins are associated with kidney stone disease and whether protein measurements improve the identification of stone formers compared with conventional risk factors (RF). Using Western blotting, we studied variations in the electrophoretic mobility patterns and relative abundances of crystallization-inhibitory proteins in urine from 50 stone-forming (SF) and 50 non-stone-forming (NS) first-degree relatives of calcium SF patients, matched by gender and age. Standard urine chemistry stone risk measurements were also made. Multivariate discriminant analysis was used to test the association of these proteins with nephrolithiasis. Differences in form and abundance of several urine proteins including inter-alpha-trypsin inhibitor (ITI), prothrombin fragment 1 (PF1), CD59, and calgranulin B (calB) were found to be associated with stone formation. By multivariate discriminant analysis, measurements of forms of PF1, ITI, and calB in men and ITI and CD59 in women, classified 84% of men and 76% of women correctly by stone status. In contrast, standard urine chemistry RF identified only 70% of men correctly and failed to distinguish female SF from NS. Thus a small subset of protein measurements distinguished SF from NS far better than conventional RF in a population of relatives of calcium SF, illustrating the significant association of these proteins with stone disease. Variations in these proteins may serve as markers of stone disease activity or vulnerability to recurrence and may provide new insights into mechanisms of stone formation.