The present studies describe the distribution of phosphatidylinositol (PI) within the membrane bilayer of the human red blood cell (RBC) as well as its transbilayer mobility. The membrane bilayer distribution was determined by measuring the hydrolysis of PI in the exterior leaflet of the RBC membrane using a PI-specific phospholipase C and by extraction of PI from the exterior leaflet using bovine serum albumin. The transbilayer mobility of PI was measured by following the fate of radiolabeled PI which was first incorporated into the outer leaflet of the RBC membrane. Our results indicate that PI is asymmetrically distributed in the membrane, with approximately 80% located in the inner and 20% in the outer leaflet of the bilayer. The rate of transbilayer mobility of PI is similar to that for certain molecular species of phosphatidylcholine and much slower than that reported for the aminophospholipids in the RBC membrane.