Serum cystatin C concentration correlates negatively with glomerular filtration rate as well as or better than that of serum creatinine, suggesting a constant formation, and elimination from extracellular fluid mainly by glomerular filtration. It is not known, however, how well the renal plasma clearance of this 13-kDa basic polypeptide matches the glomerular filtration rate. This was investigated in rats during control conditions and after reduced renal perfusion pressure. ¹²⁵I-cystatin C and an indicator for glomerular filtration (⁵¹Cr-EDTA or ¹³¹I-aprotinin) were injected intravenously. The renal accumulation and urinary excretion of the tracers were recorded in periods of 2.5 to 20.0 min. The renal plasma clearance of ¹²⁵I-cystatin C (C_cy) based on the renal content of ¹²⁵I correlated well with the glomerular filtration rate (C_cr-edta) in periods up to 6 min; i.e. C_cy=0.94 C_cr-EDTA, r=0.99. Less than 0.5% of the filtered amount appeared in the urine. During more prolonged periods, C_cy increasingly underestimated glomerular filtration rate, reaching about 0.4 C_cr-EDTA in a 20-min period. Free ¹²⁵I relative to total plasma ¹²⁵I activity increased from about 2% at 5 min to about 70% at 20 min. In nephrectomized rats, free ¹²⁵I accumulated in plasma at a slower rate, accounting for about 15% of the total activity 20 min after injection of ¹²⁵I-cystatin C. We conclude that cystatin C is (a) mainly removed from the extracellular fluid by the kidneys, (b) practically freely filtered in the glomeruli, and (c) completely absorbed and rapidly broken down by the proximal tubular cells.