Proteins in first voided urine from premature and term neonates were analyzed by two-dimensional gel electrophoresis. At 22-28 weeks of gestation, urine contained many identifiable serum proteins and the presence of an array of minor polypeptides with masses below 40 kilodaltons (kd) made the patterns quite complex. In contrast, the abundance of serum proteins in term urine was markedly decreased and the polypeptide patterns approached the relative simplicity seen in adult urine. Urine contained three major nonserum polypeptide groups at 30.5, 33-49, and 97 kd. The 97-kd species was identified as the Tamm-Horsfall glycoprotein, which is known to be derived from the renal tubule.