Large quantities of peptides (1-4 g) are excreted in human urine each day. In this study we sought to analyze how peptide excretion varies with increasing albuminuria associated with diabetes, as well as to characterize the size distribution of albumin-derived peptides in urine from volunteers without diabetes and from patients with macroalbuminuria and diabetes. We detected albumin-derived peptides by injecting tritiated albumin intravenously into human volunteers and patients with diabetes. Urine was collected after 24 hours and fractionated on a size-exclusion column. This fractionation revealed peptides with molecular weights ranging from 300 to 500 Da in volunteers without diabetes. The albumin-derived peptides were of higher molecular weight in the urine of a patient with macroalbuminuria and diabetes. The molecular-weight distribution of the peptides derived from tritiated albumin peptides was paralleled by the distribution of all protein peptides (including albumin) as determined with the use of the Biuret protein assay or absorbance at 214 nm. We determined peptide-excretion rates by filtering urine from patients with diabetes through a 10,000 Da molecular-weight-cutoff membrane and then measuring the filtrate with the use of the Biuret assay. This analysis revealed that the peptide-excretion rate increased with increasing total protein excretion, regardless of whether the patient demonstrated normoalbuminuria or microalbuminuria. Among patients with macroalbuminuria, the peptide-excretion rate leveled off and even decreased in the face of an increasing albumin concentration or protein-excretion rate. This study confirms that albumin-derived and protein-derived peptides exist at high concentrations in urine. Although peptide-excretion rates are maintained at similar levels up to macroalbuminuric states, the relative proportion of peptide excretion is significantly reduced compared with total protein.