Menkes' kinky hair syndrome (KHS) is a lethal x-linked neurodegenerative disorder of copper metabolism, with low serum copper concentrations, tissuespecific copper sequestration, and decreased activities of cuproenzymes in a number of cell types. Although liver copper accumulation is abnormal in KHS, the actual defect in hepatic copper metabolism has not been elucidated. Our studies of liver copper metabolism were conducted in the mottled (blotchy) mouse, an animal model of KHS. After implantation of central venous and biliary catheters in both blotchy and control mice, we measured biliary copper excretion, hepatic copper uptake, and tissue copper contents over an 8-h period after i.v. bolus administration of radioactive62Cu. Under the experimental conditions used, bile flow and biliary bile acid excretion were held constant, and control and blotchy hepatic64Cu concentrations were similar in the face of the expected differential in control and mutant kidney64Cu contents. Biliary excretion of radiocopper was 24.7 ± 1.5% of injected64Cu over 8 h in control animals, whereas heterozygotes excreted 6.5 ± 1.3% and a single hemizygote excreted less than 2%. The pattern of biliary copper excretion was different, with sharp increase and steady decline in control biliary64Cu excretion but consistently low excretion in mutant mice. No differences were observed in control or mutant hepatic uptake of64Cu. These data show a reduced biliary excretion of copper in the blotchy mouse, in the absence of a defect in hepatic copper uptake. We suggest that defective copper transport from hepatocyte to bile represents the hepatic expression of the mottled mutation and speculate that a similar defect occurs in human KHS. These conclusions are consistent with the hypothesis that the basic defect in mottled mutants and in KHS affects cellular copper transport or the intracellular delivery of copper to cellular transport systems.