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The purpose of this study was to determine the effect of twin-to-twin delivery interval on umbilical artery acid-base status of the second twin at birth. This was a retrospective cohort study of all live-born twins with measured acid-base status in umbilical arterial blood who were delivered after 34 weeks’ gestation from June 2003 to February 2006. Twins with any maternal or fetal complications were excluded. Subjects were divided into two groups based on the mode of delivery of the first twin: normal cephalic vaginal deliveries (n=40) or cesarean deliveries (n=67). The inter-twin differences in umbilical arterial blood pH, PCO2, PO2, and base excess in twin newborns born vaginally were significantly greater than the corresponding differences in those born by cesarean section. A significant positive correlation was found between twin-to-twin delivery interval and inter-twin difference in umbilical arterial blood pH in twin newborns born vaginally. The umbilical arterial blood pH of the second twin was less than 7.0 in 14% (2/14) in cases delivered more than 20 min after the first twin. The umbilical arterial blood gas status of the second twin worsened with increasing twin-to-twin delivery interval, and pathologic fetal acidemia (pH <7.0) might develop in the second twin when the twin-to-twin delivery interval was greater than 20 min.