AbstractWe have studied the biosynthesis of the insulin receptor in a human hepatoma cell line, HepG2. As previously reported, these cells synthesize a disulphide–bonded α2 β2 tetrameric insulin receptor. Labelling of HepG2 cells with [3H]palmitate or [3H]myristate followed by immunoprecipitation with a polyclonal antireceptor antibody revealed the incorporation of palmitate, but not myristate, into the β‐subunit and αβ‐precursor of the receptor in a hydroxylamine‐sensitive linkage. The extracellular α‐subunit was not labelled, demonstrating the specificity of incorporation. Acylation of the insulin receptor was an early event as judged by fatty acid incorporation into the αβ‐precursor and prevention by protein synthesis inhibitors. Pulse‐chase studies demonstrated the expected processing of the αβ‐precursor to mature α‐ and β‐subunits, but no evidence for preferential turnover of the fatty acid moiety was found. The site of acylation appears to be in the transmembrane or cytoplasmic domain since proteolytic treatment of intact cells produced a truncated β‐subunit still containing label. Binding studies showed that HepG2 cells contain approximately half as many insulin‐like growth factor‐1 receptors as insulin receptors, raising the possibility that this receptor may also be acylated. Indeed, immunoprecipitation with the antiinsulin receptor serum of MDCK cells expressing IGF‐1 receptors, but not insulin receptors, revealed bands corresponding to the αβ‐precursor, α‐ and β‐subunits, of which the αβ‐precursor and β‐subunits incorporated [3H]palmitate but the α‐subunit did not.