Experiments were conducted to establish an in vitro AChE inhibition test system to evaluate the potency of AChE inhibition of new chemical compounds. For a fixed time inhibition test, optimal inhibition (incubation) time to evaluate their AChE inhibition potency was 10 min. for AChE inhibitors such as DFP, DDVP, and paraoxon. The concentration of new chemical compounds with an ester group for evaluation of their inhibition potency was 10 $mu$M under 10 min. preincubation conditions. However, the stepwise inhibition test with higher concentrations seemed to be needed for other chemical compounds. For a progressive inhibition test to calculate inhibition constants such as $K_d$, $K_3$ and $K_i$, extremely low $K_d(1.3 imes10-^85.6 imes10^{-7})$ and $K_3$(0. 21-0.27 $min^{-1}$) were observed under lagged preincubation time (0.8-13.3 min) and low in¬hibitor concentrations $(1 imes10-^92 imes10-^6M)$. However, this method seemed to be useful for comparison of AChE inhibition potency among inhibitors. Differences in inhibition potency among DFP, paraoxon, and KH501 were due to the differences in $K_d$, in other words, differences in affinities between inhibitors and AChEs. Therefore, AntiChE screening should consist of two steps. The first step is to evaluate the potency of AChE inhibition based on $I_50$ valuse obtained from fixed time inhibition tests. The second step is to study inhibition patterns and characteristics of chemical compounds selected in the first step.