The liver is largely responsible for free hemoglobin uptake, but the molecular mechanism of this phenomenon has never been revealed. This paper presents the results of the study on hemoglobin binding components of the hepatocyte membrane that were purified using affinity chromatography on a hemoglobin matrix and identified by peptide mass fingerprinting. Both F1-ATPase alpha and beta subunits were retrieved. The binding was confirmed via an intrinsic fluorescence quenching study using a purified recombinant F1-ATPase beta subunit, and the dissociation constant for the complex was estimated from the saturation binding curve (Kd = 7.5 x 10(-7) M). The results indicate that haemoglobin binds to hepatocyte ectopic F1-ATPase. We suggested the plausible role of the receptor in endocytosis of haemoglobin by the hepatocyte.