The purpose of this investigation was to determine whether acetylcholinesterase (AChE) can be used as a marker of cell aging in human red blood cells (RBCs). This study used consented subjects; both males and females in an age range of 21–42 years. The blood samples (8–9 mL) were drawn in tubes containing sodium heparin or EDTA as anticoagulants. To avoid contamination with other cells, (lymphocytes, monocytes and reticulocytes), RBCs were purified (PRBC) by Hypaque-Ficoll gradient technique. The PRBCs were subfractionated into young (y) (1.08–1.09), mid (m) (1.09–1.11) and old (o) (1.11–1.12) percoll density ( g mL ) fractions using a discontinous percoll gradient. The mean ± 1 SD AChE per gram hemoglobin ( U g Hgb ) activities in whole blood (WB) purified human red blood cells (PRBCs), young human red blood cells (y-RBCs), mid age human red blood cells (m-RBCs) and old human red blood cells (o-RBCs) were 27.4 ± 2.98, 26.0 ± 2.33, 25.5 ± 1.64, 20.3 ± 3.84, 14.6 ± 3.42 in males and 26.3 ± 4.44, 24.8 ± 4.83, 26.4 ± 4.59, 24.0 ± 5.50 and 12.4 ± 7.09 in females respectively. Although there was variation in the data, the results indicated that old human red blood cells showed significantly (p < .05) lower AChE activity compared to young human red blood cells of both sexes. These preliminary but novel observations suggest that AChE can be an excellent enzymatic marker for RBC aging in man.