In vitro studies suggest a free bilirubin (B(F)) concentration in the range of 71-770 nmol/L can induce neurotoxicity. In vivo data regarding central nervous system (CNS) B(F) levels have not been determined. We calculated in vivo CNS B(F) levels in Gunn rat pups (15-19 d old; heterozygous nonjaundiced Gunn rats (J/j) and homozygous jaundiced Gunn rats (j/j); saline or sulfadimethoxine treated) based on 1) total brain bilirubin (TBB) content, 2) brain albumin level, 3) CNS bilirubin binding capacity attributable to brain albumin determined using an ultrafiltration technique, and 4) published Gunn rat albumin-bilirubin binding constants (k). Gunn rat brain bilirubin binding capacity was approximately 22 x 10(-3) micromol/g, of which two thirds was accounted for by brain albumin. Using a Gunn rat pup in vivo, k of 9.2 L/micromol, calculated CNS B(F) levels ranged from 72 to 112 nmol/L [95% confidence interval (CI)] in saline and from 59 to 156 nmol/L (95% CI) in sulfadimethoxine-treated J/j pups. These animals demonstrated no neurobehavioral abnormalities and normal cerebellar weight. Calculated CNS B(F) levels were severalfold higher (p < 0.001) in saline (95% CI: 556-1110 nmol/L) and sulfadimethoxine-treated (95% CI: 3461-8985 nmol/L) j/j pups; the former evidenced reduced cerebellar weight; the latter both reduced cerebellar weight and acute neurobehavioral abnormalities. We conclude that calculated CNS B(F) values in j/j pups are substantially higher than those in J/j animals. Given the absence of CNS abnormalities in J/j pups, the presence of such in j/j animals, and the CNS B(F) levels in these groups, we speculate that the CNS B(F) neurotoxicity threshold in vivo is subsumed within the range (71-770 nmol/L) reported in vitro.