Bilirubin-IX alpha photooxidation products were detected by high performance liquid chromatography in the urine of neonates undergoing phototherapy for hyperbilirubinemia. The in vivo photoproducts were identified by chromatographic comparison with authentic synthetic standards using two complementary methods. Bilirubin photooxidation products were not detected in urine from jaundiced infants not receiving phototherapy. The specific photoproducts identified in the urine include propentdyopents, hematinic acid imide and its hydrolysis product (3-carboxy-2-methyl-2-hexenedioic acid), and the hydrolysis product (2-vinyl-3-methyl-maleic acid) of methylvinylmaleimide. Their total urinary concentrations were low (0.2-0.9 mg/dl) during phototherapy. These observations show that photooxidation of bilirubin clearly does occur during phototherapy. They are consistent with the view that, although photooxidation is not the major photochemical event associated with phototherapy, it can and clearly does occur concurrently with photoisomerization.