After birth, constriction of the full-term ductus arteriosus produces ischemic hypoxia, caspase activation, DNA fragmentation (Ͼ70% of cell nuclei are positive by the terminal deoxynucleotidyl transferase nick-end labeling [TUNEL] technique), and permanent ductus closure. In contrast, the preterm ductus frequently fails to develop these changes. We used the TUNEL technique to examine rings of fetal ductus arteriosus (incubated for 24 h at different oxygen and glucose concentrations) to determine the roles of 1) constriction and shortening, 2) hypoxia, and 3) hypoglycemia in producing cell death. Under controlled conditions, late-gestation ductus rings had a low rate of TUNELpositive staining (0.6 Ϯ 0.9%) that did not change during muscle shortening. Although hypoxia (6.9 Ϯ 3.5%) and hypoglycemia (2.4 Ϯ 1.9%) increased the incidence of TUNEL-positive staining, only the combination of hypoxia-plus-hypoglycemia increased the incidence to the range found in vivo (83 Ϯ 9.5%). The combination of hypoxia-plus-hypoglycemia was associated with an oligonucleosomal pattern of DNA fragmentation. Under the same experimental conditions, the preterm ductus was capable of developing a similar degree of TUNEL-positive staining as found at term. Although caspase-3 and caspase-7 were activated in rings exposed to hypoxia-plus-hypoglycemia, a nonselective caspase inhibitor, Z-VAD.FMK (which inhibited caspase-3 and caspase-7 cleavage in the rings), did not diminish the degree of TUNEL-positive staining. We hypothesize that the preterm ductus is capable of developing an extensive degree of cell death, if it can develop the same degree of hypoxia and hypoglycemia found in the full-term newborn ductus. We also hypothesize that cell death in the ductus wall may involve pathways that are not dependent on caspase-3 or -7 activation. In the full-term neonate, closure of the ductus arteriosus occurs in two phases: first, smooth muscle constriction reduces the size of the ductus lumen during the first hours after birth; this is followed by a second phase, involving loss of ductus responsiveness to vasoactive stimuli and anatomic remodeling of the ductus wall (1). Death of smooth muscle cells in the inner ductus muscle media plays an essential role in the loss of vasoreactivity and the anatomic remodeling (2, 3). Within 24 h of birth, more than 70% of the smooth muscle cells in the inner muscle media have evidence of DNA fragmentation and cell death (demonstrated by the in situ TUNEL technique) (4).The events that are responsible for ductus smooth muscle cell death have recently been described. During postnatal constriction, blood flowing through the vasa vasorum that penetrate and run through the outer muscle media of the ductus wall is obstructed. This produces a zone of profound hypoxia (tissue oxygen concentration Ͻ0.4% oxygen) and nutrient ischemia in the ductus muscle media (4). Ductus constriction, and the profound ischemia that accompanies it, are required before cell death and remodeling take place (2, 5-7)Several of the features of ...