Neonatal jaundice is usually treated with phototherapy that converts bilirubin to more polar stereoisomers. These should theoretically be less able to cross the blood-brain barrier. The rates of photoisomer formation and concentrations accumulating in the circulation may have a bearing on the risk of kernicterus. The purpose of this study was to determine the rate of appearance of the major 4Z, 15E photoisomer of bilirubin during the early stages of phototherapy. Twenty jaundiced neonates were treated with phototherapy, and blood samples were drawn before and at approximately 15, 30, 60, and 120 min (10 infants) or at approximately 15, 60, 120, and 240 min (10 infants) after beginning phototherapy. Blood samples were analyzed for total serum bilirubin (TSB) and the 4Z, 15E photoisomer of bilirubin. Significant (p<0.0001) formation of the 4Z, 15E photoisomer was detectable within 15 min. The change in TSB from time 0 was insignificant at 120 min but reached significance at 240 min (p<0.001). The 4Z, 15E bilirubin constituted up to 20-25% of TSB at 2 h and may not have peaked by 4 h. Further studies are needed to determine whether this early shift in balance between bilirubin isomers with different polarities may impact the risk of bilirubin encephalopathy even before TSB starts to fall.