Angiotensin I-converting enzyme (ACE) isoforms in urine from healthy and mildly hypertensive untreated patients have been described in the literature. Healthy subjects have high- and low-molecular-weight ACEs (170 and 65 kDa), whereas mildly hypertensive untreated patients have only low-molecular-weight ACEs (90 and 65 kDa), both of which resemble ACE from the N-terminal domain. Previous studies have shown that ACE is regulated during development, and renal tubules of premature human infants are not completely mature, given that nephrogenesis is not complete until the 36th week of gestation. The aim of the present study was to purify and characterize ACE isoforms from urine of premature and full-term infants and to detect the presence of the N-domain form of ACE during prenatal development. Urine from premature and full-term infants was concentrated in an Amicon concentrator, dialyzed in the same equipment against 50 mmol/L Tris-HCl buffer (pH 8.0) that contained 150 mmol/L NaCl, and submitted to gel filtration on an AcA-34 column equilibrated with the buffer described above. Two peaks (P1 and P2 for premature infants; TP1 and TP2 for full-term infants) with ACE activity on hippuryl-His-Leu (K(m), 3 mmol/L) were detected. All enzymes were Cl(-) dependent and inhibited by captopril and EDTA. The peptides angiotensin-(1-7) and N-acetyl-Ser-Asp-Lys-Pro, described as specific for N-domain ACE, were hydrolyzed by P2 and TP2, which suggests that both enzymes are N-domain ACE. In premature infants, P1 activity with hippuryl-His-Leu was 12-fold lower than P2 activity, but in full-term infants, the difference between TP1 and TP2 was 1.6-fold. Chromatography profiles of urine from premature infants were analyzed on days 1, 3, 7, 14, 21, and 30 after birth. The P1 of ACE was detected around the 21st and 30th days, whereas P2 was detected from day 1. These results suggest that ACE activity is related to renal development and that N-domain ACE as well as full-length ACE is present in urine from premature infants. This may indicate that healthy subjects produce and secrete the N-domain form of ACE even before term development.