OBJECTIVE To determine the effect of phototherapy on serum conjugated bilirubin fractions, an index of bilirubin conjugation, in hyperbilirubinemic neonates. METHODS Serum was sampled from 21 jaundiced (serum diazo total bilirubin > or = 274 micromol/l), term, otherwise healthy neonates prior to starting phototherapy, and 24 h after the commencement of treatment. Alkaline methanolysis followed by reverse-phase, high-performance liquid chromatography, specific for determination of unconjugated bilirubin and the monoconjugated and diconjugated fractions of total conjugated bilirubin in serum, was used for the analysis. Prephototherapy values were compared to those at 24 h. RESULTS Serum total bilirubin and total conjugated bilirubin values decreased during the study period, from 220 (211-239) to 177 (157-213) micromol/l (median (25-75% range)) p = 0.001, for the former, and from 1.4 (0.87-1.57) to 0.93 (0.69-1.84) micromol/l, p = 0.005, for the latter. These parameters decreased by a similar percentage (-16.6+/-14.8% and - 14.0+/-23.4%, respectively; p > 0.05). Both monoconjugated and diconjugated bilirubin, calculated as a percentage of total conjugated bilirubin, remained constant over the study period (88.2 (72.2-96.4)% before phototherapy and 92.5 (87.3-96.8)% after 24 h, p > 0.05, for monoconjugated bilirubin, and 11.8 (3.6-27.8)% and 7.5 (3.2-12.7)%, respectively, p > 0.05, for diconjugated bilirubin). CONCLUSIONS Serum total conjugated bilirubin values decreased in parallel to serum total bilirubin levels during phototherapy, maintaining a constant relationship between these two parameters. The ratios of monoconjugated and diconjugated bilirubin to total conjugated bilirubin remained constant. These findings imply that phototherapy does not alter bilirubin conjugation in hyperbilirubinemic neonates.