This work addresses the kinetic analysis of the interaction of methotrexate (MTX) with human erythrocyte membrane-bound acetylcholinesterase (AChE, EC 3.1.1.7). It was found that the MTX effect was independent of time of incubation with AChE before the addition of substrate which proves its reversible action. The IC50 was determined, by three methods, to be 0.73 mM. The Michaelis-Menten constant (Ks) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.13 mM in the control system, a value decreased by 30-61% in the MTX treated systems. The Vmax was 1.27 mumole/min/mg protein for the control system while it was decreased by 44-77% in the MTX treated systems. The Lineweaver-Burk plot Dixon Plot, and their secondary replots indicated that the nature of the inhibition was of the linear mixed type, i.e. uncompetitive and noncompetitive. The values of Ki(slope) and Ki(intercept) were estimated as 1.67 and 0.34 mM, respectively.