Obstructive nephropathy, the primary cause of renal insufficiency in infants, is characterized by progressive renal apoptosis. Ceramide is a sphingolipid known to stimulate apoptosis in the kidney. We investigated the effects of unilateral ureteral obstruction (UUO) on endogenous renal ceramide content and apoptosis in neonatal and adult rats. Animals were subjected to UUO or sham operation on the first day of life and were studied 3-28 days later. Adult rats were similarly treated and then studied 3 or 14 days later. In additional neonatal rats, the obstruction was removed after 5 days, with study at 14 or 28 days. Renal ceramide content was measured by diacylglycerol kinase assay, and apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end-labeling technique. Renal ceramide content was 50-fold higher in the 3-day neonatal compared with the adult kidney and 10-fold higher in the 7-day neonatal compared with the adult kidney, but there was no additional effect of UUO on ceramide content at these ages. However, after 14 or 28 days UUO in the neonate, renal ceramide was elevated compared with sham or intact opposite kidneys, and renal apoptosis was directly related to ceramide content (r = 0.99, P < 0.001). Moreover, renal ceramide was reduced by relief of obstruction (P < 0.05). There was less apoptosis in the obstructed kidney of the adult than the neonate, and UUO had no effect on ceramide content at 14 days in the adult. We conclude that prolonged UUO (at least 14 days duration) increases endogenous renal ceramide in the neonatal but not the adult rat. It is likely that this contributes to the prolonged renal apoptotic response of the neonatal obstructed kidney.