Recent studies have shown that [3H]palmitic acid strongly labels both glycosylated forms (gp22 and gp24) of the signal-initiating cell surface glycoprotein CD9. We performed a two-dimensional limited proteolysis analysis with Staphylococcus aureus V8 proteinase in order to localize the palmitylation sites to final peptides on both glycosylated forms of CD9. Analysis of [3H]leucine- and [3H]amino acid mixture-labeled gp22 delineated 4 final peptides of 11, 8, 7 and 4 kDa. gp24 produced a similar pattern with the exception that the 11 kDa peptide was replaced by an N-glycosylated 13 kDa peptide. Since all four final peptides (total molecular mass of 30/32 kDa) could not be accommodated by a parent molecule of 22/24 kDa, it is likely that one of the final peptide coexists in two differently modified states. Palmitic acid labeled the 11 kDa/13 kDa final peptides, and the 7 kDa final peptide, with equal intensity, but was not incorporated into the 4 kDa final peptide, demonstrating that fatty acid is ligated in two distinct regions of the molecule. The 8 kDa final peptide was strongly labeled by [3H]palmitic acid, but only weakly by [3H]leucine. We present evidence that this peptide is derived by further acylation of the region defined by the 7 kDa peptide, and that this occurs in only 15% of the molecules. Palmitic acid is turned over faster at these additional sites, indicating that they may be more accessible to membrane transacylases. Proteolysis of CD9 on the intact cell with papain enabled the highly acylated region to be localized to a membrane-associated fragment which contains the binding site for the agonistic monoclonal antibody 50H.19. The co-localization of a functional domain with a region of variable acylation suggests that acylation events may play a role in the transduction of the signal initiated by interaction of the antibody with CD9.