The glycosaminoglycans (GAG) isolated from pooled urine of preterm newborns were fractionated by stepwise elution from a Dowex 1-X2 column. Analytical reactions, cellulose acetate electrophoresis and enzymatic digestions with chondroitinases and testicular hyaluronidase were performed on each fraction. Chondroitin-4-sulfate and chondroitin-6-sulfate constitute about 60% of urinary GAG; heparan sulfate amounts to about 20%, while chondroitin represents 12% of total GAG; dermatan sulfate, hyaluronic acid and keratan sulfate are present in small traces. Approximately 30% of the total is constituted by nonsulfated GAG.