L-beta-Aspartyl-L-lysine and L-gamma-glutamyl-L-ornithine were isolated from pooled normal human urine and each peptide was shown to be identical with the authentic peptides. The concentrations of these dipeptides in the urine of individual subjects were determined directly by using a new buffer sequence on a standard ion exchange chromatographic amino acid analyzer with a sensitivity of 10(-10) mol. In urine from normal subjects ranging in age from 12 to 64 years, mean values of 1.47 mumol/g of creatinine of L-gamma-glutamyl-L-ornithine and 8,24 mumol/g of creatinine of L-beta-aspartyl-L-lysine were found. The urine of children under 10 years of age contained, relative to creatinine excretion, more L-beta-aspartyl-L-lysine and L-gamma-glutamyl-L-ornithine than that of older children and adults. All urines contained substantially larger concentrations of L-beta-aspartyl-L-lysine than of L-gamma-glutamyl-L-ornithine. Both peptides were found in urine collected after 21 hr of fasting in lower concentrations than found in urine from nonfasting subjects. The urinary concentrations of both peptides did not appear to be influenced by race or sex.