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Bilirubin and its photoisomers in the biological fluids of a hyperbilirubinaemic newborn infant before and during phototherapy were analyzed by a recently improved HPLC method. In the serum, the percentages of (EZ)- and (ZE)-bilirubin in the total bilirubin concentration before phototherapy were approximately 10% and on average increased over 1.5-fold at 2 h after initiation of phototherapy. The percentage of the (EZ)-cyclobilirubin in the serum bilirubin was under 1%. In the bile, the mean concentration of (ZZ)-bilirubin, derived mainly from (ZE)-bilirubin, nearly tripled during phototherapy. The (EZ)-cyclobilirubin concentration in the bile was very low before phototherapy, increased nearly ten-fold at 3 h after initiation of phototherapy, and was 5- to 6-fold as high as that of (ZZ)-bilirubin. In the urine, upon exposure to light, the urinary concentration of (EZ)-cyclobilirubin is apparently equivalent to half of the biliary concentration of (ZZ)-bilirubin and one-fifth of that of (EZ)-cyclobilirubin. It was concluded that during phototherapy of neonatal hyperbilirubinaemia the structural photoisomer [(EZ)-cyclobilirubin] predominates considerably over the geometric photoisomer [(ZE)-bilirubin].