CD9 is a signal-initiating glycoprotein of uncertain membrane insertion which contains more than one locus of acylation and is distinguished by being the major acylatable platelet protein. The N-terminus of CD9 is blocked to Edman degradation. We investigated whether [3H]myristic acid could be incorporated into CD9, whether that incorporation occurred via an amide linkage, and whether myristate and palmitate were differentially incorporated into the two domains. Pulse-labeling studies, performed on the human osteogenic sarcoma cell line SKOSC which expresses 22 and 24 kDa variants of CD9 demonstrated that the respective precursors of 20.5 and 23 kDa were not radiolabeled by either [3H]myristic acid or [3H]palmitic acid, but that both fatty acids could be ligated to CD9 during the later stages of protein maturation. The failure to incorporate myristic acid cotranslationally suggest that CD9 does not contain amino-terminal amide-bonded myristic acid. Incorporation of radiolabel from both fatty acids proceeded very rapidly and could be visualized after a 10 s pulse. Although myristic acid was partially metabolized into palmitic acid, incorporation of authentic [3H]myristate into CD9 could be demonstrated. The myristic acid bonds were shown to be as sensitive to hydroxylamine treatment as those linking palmitate. Both fatty acids were also incorporated into CD9 in hydroxylamine-sensitive bonds in the presence of cycloheximide, reaching 30-40% of the levels in untreated controls. The sensitivity of myristate ligands to hydroxylamine demonstrates that this fatty acid is not linked via amide, but rather via ester bonds. The sensitivity of [3H]myristate and [3H]palmitate bonds to 2-mercaptoethanol further suggests that either fatty acid is linked via thioester rather than hydroxyester bonds to each domain on CD9. Limited proteolysis analysis with Staphylococcus aureus V8 proteinase of CD9, labeled in the absence or presence of cycloheximide, showed that [3H]myristic acid and [3H]palmitic acid labeled identical peptides, and to the same extent, suggesting that myristate is an alternative substrate for the transacylase(s) involved.