We report here for the first time that ASGP-Rs expressed in the human hepatoma cell lines HuH-7 and HepG2 are fatty acylated. Cells were metabolically labeled with [3H]palmitate and active ASGP-Rs, which contain two subunits (HHL1 and HHL2), were purified by affinity chromatography and subjected to nonreducing SDS-PAGE and fluorography. [3H]Palmitate was covalently incorporated into both HHL1 and HHL2. When gel slices containing HHL1/HHL2 were treated at neutral pH with 1 M hydroxylamine, but not 1 M Tris, > 95% of the radioactivity was removed, indicating that the attachment of palmitate to ASGP-Rs is to cysteines. Furthermore, the same mild hydroxylamine treatment caused partial ASGP-R inactivation; 50-70% of receptors corresponding to the previously characterized State 2 ASGP-Rs were inactivated. We conclude that both HHL1 and HHL2 are covalently modified by fatty acylation, which may regulate the ligand-binding activity of human State 2 ASGP-Rs. We propose that fatty acylation/deacylation of cytoplasmic domains is a general mechanism by which extracellular ligand-binding activity of oligomeric transmembrane receptors can be regulated.