The reaction of the reversible acid anhydride, 3,4,5,6-tetrahydrophthalic anhydride with human erythrocyte membranes is shown to cause solubilization of up to 85% of the membrane protein and approximately 40% of the carbohydrate and phospholipid of the membrane. Electrophoretic analysis using polyacrylamide gels containing soldium dodecyl sulfate shows that, at low levels of chemical modification, the proteins solubilized are predominantly peripheral (bands 1, 2, 4, 5, and 6), while at higher levels, integral proteins (band 3 and periodic acid-Schiff bands 1 and 3) are also solubilized. Associated with the chemical modification of amino groups are decreases in the acetylcholinesterase activity and the turbidity of the membrane preparation. The removal of the 3,4,5,6-tetrahydrophthaloyl groups is achieved by incubation at pH 6 for 36 h, and leads to a complete recovery of acetylcholinesterase activity, but little change in turbidity. By reversing the supernatant and pellet fractions of modified ghost samples separately, it was found that the majority of the acetylcholinesterase remained associated with the pellet fraction. Gel filtration and sucrose density gradient experiments were used to determine the molecular nature of the species present in solution and the results show that, after reversal of chemical modification, there is substantial reassociation of the membrane protein with the membrane phospholipid.