The transmembrane glycoprotein HEF and its acylation deficient mutant M1 were expressed in Sf9 insect cells infected with recombinant baculovirus and in CV1 mammalian cells using the vaccinia T7 system. In insect cells (Sf9), both wild type HEF and HEF(M1) are synthesized in their precursor form HEF0, which appears as a double band in SDS gels. Digestion with glycopeptidase F and endoglycosidase H reveals that the larger 84-kDa form is modified by the attachment of unprocessed carbohydrates of the high mannose type whereas the smaller 76-kDa form is non-glycosylated. As revealed by in vitro labeling experiments with palmitic acid another modification of HEF is the attachment of a long chain fatty acid to cysteine residue Cys-652 which is located at the internal border of the cytoplasmic membrane. After labeling with [3H]palmitic acid in both systems only HEF(WT) is acylated, whereas HEF(M1) is not. High performance liquid chromatography analysis of the fatty acids bound to HEF(WT) expressed in Sf9 insect cells reveals nearly 80% of palmitic acid. In contrast to this finding, the acylation pattern of HEF expressed in CV1 cells shows nearly the same amounts of stearic and palmitic acid (40%). Since the interconversion of the input [3H]palmitic acid to stearic acid is even lower in CV1 cells than in insect cells, it follows that only HEF expressed in mammalian, but not in insect cells selects for stearic acid during its biosynthetic acylation. We extended our study to acylation of endogenous proteins in Sf9 cells. In finding only palmitate linked to protein we present evidence that, in contrast to mammalian cells, insect cells (Sf9) cannot transfer stearic acid to polypeptide. This finding favors the hypothesis of enzymatic acylation over non-enzymatic mechanisms of acyl transfer to protein.