Several urinary macromolecules are known to modulate calcium oxalate (CaOx) crystallization. One of these is urinary bikunin, the light chain of inter-alpha-inhibitor. Bikunin has been demonstrated to be an efficient inhibitor of CaOx crystal growth; however, its inhibitory activity against other events in CaOx crystallization has not been fully investigated. To assess the potential of urinary bikunin as an effective inhibitor, its effects on CaOx crystal nucleation and aggregation were evaluated. Nucleation and aggregation of CaOx crystals were studied by measuring turbidity at 620 nm. In the nucleation assay, crystallization was induced by mixing calcium chloride and sodium oxalate, at final concentrations of 3 and 0.5 mM, respectively. Both solutions were buffered with 0.05 M Tris, 0.15 M NaCl, pH 6.5. Nucleation measurements were performed at 37 degrees C, with stirring at 800 rpm. Inhibition of nucleation was estimated by comparing the induction time in the presence of the inhibitor with control values. In the aggregation assay, the optical density of the solution containing CaOx monohydrate crystals was monitored. Inhibition of aggregation was evaluated by comparing the turbidity slope in the presence of the inhibitor with control values. The data showed that urinary bikunin, at concentrations of 2.5 to 20 microg/ml, retarded crystal nucleation by 67 to 58% and inhibited crystal aggregation by 59 to 80%. According to these results, it seems that urinary bikunin is an efficient inhibitor of crystal nucleation and aggregation. Its presence in the kidneys and urine may protect subjects against CaOx crystallization and kidney stone formation.