On routine blood screens, persistent conjugated hyperbilirubinemia was discovered in two groups of closely related adult female golden lion tamarins (Leontopithecus rosalia rosalia, n = 8). Bromosulfophthalein (BSP) retention tests were performed on four hyperbilirubinemic and three control tamarins. BSP excretion was delayed in hyperbilirubinemic tamarins as compared with controls. Grossly, liver of affected tamarins was dark brown to black, with a prominent reticulated pattern. Histologic examination revealed abundant intrahepatic pigment, primarily in a centrilobular and midzonal distribution. Most of the pigment did not react with Perls' Prussian blue method for iron, Hall's method for bilirubin, or the Armed Forces Institute of Pathology acid-fast method for lipofuscin but was positive with Fontana and lipofuscin-ferric ferricyanide reduction techniques. Liver from control golden lion tamarins had intrahepatocellular Perls' iron-positive pigment diffusely throughout the lobule with a small amount of Fontana method-positive pigment. Ultrastructurally, hepatocytes from a hyperbilirubinemic tamarin contained pleomorphic electron-dense structures within lysosomes. Transport studies demonstrated secretion of fluorescein isothiocyanate-labeled glycocholic acid, a fluorescent bile acid analog, into bile canaliculi and no secretion of carboxydichlorofluorescein diacetate, a non-bile acid organic anion, by liver from a hyperbilirubinemic tamarin. In contrast, control liver secreted carboxydichlorofluorescein diacetate readily into bile canaliculi. The clinicopathologic presentation of this syndrome in golden lion tamarins is similar to that described for Dubin-Johnson syndrome of human beings.