In the present study, the inhibitory mechanisms and effects of a synthetic phenazine dye, safranin O (SO) on human plasma butyrylcholinesterase (BChE), human erythrocyte acetylcholinesterase (AChE) and recombinant BChE mutants were investigated. Kinetic studies showed the following information: SO leaded to linear competitive inhibition of human plasma BChE with K-i = 0.44 +/- 0.085 mu M; alpha = infinity. It acted as a hyperbolic noncompetitive inhibitor of human erythrocyte AChE with K-i = 0.69 +/- 0.13; alpha = 1;beta = 0.08 +/- 0.02. On the other hand, the inhibitory effects of SO on two BChE mutants, where A328 was modified to either F or Y, revealed differences in terms of inhibitory patterns and K-i values, compared to the obtained results with recombinant wild type BChE. SO was found to act as a linear competitive inhibitor of A328F and A328Y BChE mutants. Compared to recombinant wild type BChE, A328Y and A328F BChE mutants caused a 4 and 10-fold decrease in K-i value for SO, respectively. These findings were supported by molecular modelling studies. In conclusion, SO is a potent inhibitor of human cholinesterases and may be useful in the design and development of new drugs for the treatment of AD.