The aim of this study was to identify a specific substance [a Low-Molecular-Weight Chromium binding substance (LMW Cr)] in normal urine and also to clarify the various factors affecting chromium to bind this substance in urine. The results obtained were as follows: 1. Normal human and rat urines contained unsaturated LMW Cr similar to that found in the liver or other organs of rabbits and dogs. The amount of chromium bound in vitro to the unsaturated LMW Cr in normal human urine was 14.81 +/- 1.84 microgram/ml urine when urine was incubated with trivalent chromium for 30 minutes at 37 degrees C. 2. Higher amounts of chromium were bound to LMW Cr in normal urine a 37 degrees C than at 24 degrees C, in acidic state of urine than in alkaline state, and when trivalent chromium was added to the incubation mixture than when hexavalent chromium was added. Chromium contained in LMW Cr was supposed to be trivalent. Furthermore, boiling urine at 100 degrees C for 10 minutes did not reduce the chromium binding capacity of LMW Cr in urine. These results indicate that LMW Cr in urine binds chromium by a chemical reaction. 3. Amounts of chromium bound to LMW Cr in urine obtained 3 hrs after intraperitoneal injection of 200 mumole K2Cr2O7 to a rat were much higher than amounts of chromium bound in vitro to LMW Cr in normal urine during 3 hrs incubation of the urine with K2Cr2O7. This suggests that most of excessively absorbed chromium is excreted into urine after being bound to LMW Cr in organs. 4. Urine from patients with chronic nephritis excreted a part of added chromium bound to high molecular weight fractions corresponding to peaks of UV absorption.