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 to 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 stone forming 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 males, and ITI and CD59 in females, classified 84% of males and 76% of females correctly by stone status. In contrast, standard urine chemistry RF identified only 70% of males 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 stone formers, 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.