We attempted to replace the myo-inositol in cellular inositol phosphatides with 5-deoxy-myo-inositol to evaluate the role of inositol 1,4,5-trisphosphate as a second messenger. This analog, lacking a 5-hydroxyl, might be incorporated into 5-deoxyphosphatidylinositol and converted to the corresponding phosphatidylcyclitol 4-phosphate but could not be converted to phosphatidylinositol 4,5-diphosphate, the precursor of the second messenger molecule inositol 1,4,5-trisphosphate. We synthesized 5-deoxy-myo-inositol and found that this analog does not replace myo-inositol as an essential growth factor for essential fatty acid deficient HSDM1C1 mouse fibrosarcoma cells. Furthermore, [5-3H]-5-deoxy-myo-inositol was neither incorporated into the phospholipids nor accumulated in the cytoplasm of these cells. It appears that this cell line has a specific myo-inositol uptake system that excludes a potentially harmful analog of inositol.