CD 109 is a 175 kDa glycoprotein found on platelets, endothelial cells and some cell lines. Alloantibodies to Gov-a/b antigens on CD 109 are implicated in neonatal alloimmune thrombocytopenia and post-transfusion purpura. CD 109 on activated T-cells is associated with helper, but not cytotoxic/suppressor cells. The restricted distribution of CD 109 on platelets, endothelial cells and activated T-cells suggests a role for this protein in cell adhesion. On endothelial cells, activated T-cells, and tumor cell lines, CD109 is reported to have a glycosylphosphatidylinositol (GPI) lipid anchor defined by release of CD 109 using Pi-specific phospholipase C (PI-PLC). In contrast, using '"I-labelled cells, we found CD 109 on human platelets to be only partially sensitive to PI-PLC (0.1 to 4.0 U/ mL. B. thuringiensis and B. cereus) (maximal 54% release, 1.0 U/mL). Inhibition of PIPLC by a membrane component was not a factor as only 58% of CD109 was converted to the hydrophilic form when membranes were solubilized with Triton X-114 and the hydrophobic fraction treated with PI-PLC, suggesting that alternate forms of the anchor exists on platelets. However, temperature-dependent solubilization in Triton X-100 and complete extraction from the membrane using octylglucoside indicated that platelet CD 109 resides in a cholesterol-rich membrane fraction characteristic of many GPI-anchored proteins. Since acylation of the inositol ring can interfere with the function of PI-PLC, the resistance to PI-PLC cleavage was investigated using human serum as a source of GPIPLD, an enzyme capable of cleaving all GPI-anchored proteins. Solubilized CD 109 was completely converted to the hydrophilic form by serum GPI-PLD (>95%) with inhibition by the specific GPI-PLD inhibitor, phenanthroline. Specificity of the reaction was confirmed using purified GPI-PLD, indicating that all platelet CD109 is GPI-anchored, of which half is palmitoylated on the inositol ring. Similar results were found for CD109 anchors on the megakaryocytic cell lines, MEG-01 and DAMI. Human umbilical vein endothelial cell CD 109 was 100% PI-PLC sensitive, indicating that alternate CD 109 anchoring is unique to cells of megakaryocytic lineage. In conrast, GPI-anchored CD59 was completely sensitive to PI-PLC on all cells tested, suggesting that platelet CD 109 is uniquely targeted to alternate anchoring which may play a role in the function of this protein.