The excretion of serum albumin in the urine is considered the net result of renal glomerular filtration and tubular uptake. During routine experiments, we observed that a batch of tritium-labeled albumin yielded anomalous results, being excreted in the urine of isolated perfused kidneys at 10 times the rate of normal tritiated albumin. This anomalous albumin, when simultaneously studied with normal carbon 14-labeled albumin, exhibited 10 times greater excretion than normal [(14)C]albumin. Anomalous albumin could not be reversed to normal albumin by means of conditioning with blood. In vivo clearances of anomalous albumin could not be quantitated because anomalous albumin is degraded during circulation. Anomalous albumin appeared to have the same molecular size (as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, capillary electrophoresis, and gel chromatography) and isoelectric-point profile (2-dimensional electrophresis) as normal albumin. Normal albumin could be transformed to anomalous albumin with alkali/heat treatment. Reverse-phase high-pressure liquid chromatography analysis of fragments from tryptic digests of anomalous albumin, alkali/heat-treated albumin, and normal albumin suggest that anomalous albumin and alkali/heat-treated albumin have altered tertiary structure, possibly as a result of denaturation and disulfide exchange. These studies show that the tertiary structure of albumin, beyond simple size and charge, is a critical determinant for albumin processing by the kidney and suggest that a specific albumin-recognition event by the kidneys is critical to normal renal handling of albumin.