2-Palmitoylation of the inositol residue occurs during biosynthesis of glycosylphosphatidylinositol (GPI) anchors, but the enzymology of this step has been enigmatic, With endogenously synthesized glucosamine-PI (GlcN-PI; a GPI intermediate), a CoA-dependent palmitoyl-CoA-independent acyl-transfer activity (AT-1) has been reported in rodent preparations, In contrast, a palmitoyl-CoA-dependent GlcN-PI acyltransferase activity (AT-2) was reported in both rodent and yeast preparations with a novel water-soluble dioctanoyl GlcN-PI analogue, GlcN-PI(Cg), We report that AT-1, as well as AT-2, can be detected in rodent microsomes with GlcN-PI(C8), thus demonstrating the coexistence of these activities in a single membrane preparation and the general utility of GlcN-PI(C8) for studying the GPI pathway, Unexpectedly, AT-2 was peripherally associated with microsomes, a property atypical for GPI biosynthetic enzymes.