(1) The levels of glycolytic intermediates in the brain tissue of 15-day-old homozygous and heterozygous Gunn rats were determined with or without novobiocin treatment in vivo, and the inhibitory effects of bilirubin on the glycolytic activity and the key glycolytic enzyme activities were investigated in vitro. (2) the pattern of glycolytic intermediate levels in the non-treated homozygotes compared with those in the non-treated heterozygotes showed a significant increase of G6P and F6P, and decrease of GAP plus DAP. Enhancement of bilirubin concentration in the brain tissue of homozygotes by novobiocin treatment brought about a significant decrease of FDP, 3PG, Pyr and Lac, and a slight increase of G6P compared with the respective values on the treated heterozygotes. (3) the overall glycolytic activity with 2mM glucose as a substrate was significantly inhibited in vitro by 100 muM bilirubin at 3-4, 20 and 600muM NAD concentration, whereas with 0.15 mM glucose the inhibition was observed only at a concentration of 3-4muM NAD. The inhibitory effect of bilirubin (120-140 muM) on lactate formation with FDP as a substrate was also found at three different NAD concentrations. And the marked inhibition of PFK activity by bilirubin was observed at physiological concentrations of ATP and F6P. (4) The present findings suggest that reduced rates of glycolysis might be involved in bilirubin encephalopathy.