Force spectroscopy between a single acetylcholinesterase (AChE) molecule and its natural substrates was performed, and the effects of inhibitors and reactivators on the force spectrum were studied with atomic force microscopy (AFM). The force spectrum between normal AChE and its substrates had its special shape. Inhibitors, which inhibit AChE by occupying the active center of the enzyme, could change the force spectrum shape noticeably. Reactivators, which reactivate the inhibited AChE by pulling the inhibitor off the active center of the enzyme, could make the normal shape of force spectrum reappear. This meant the shape features of the force spectrum could be used as a good index to observe the time course of the interactions between a single AChE molecule and its special inhibitors and reactivators in real time. The results of the real-time observation demonstrated that the inhibition times of soman and sarin on AChE were longer than 2 h and that of eserine, a reversible inhibitor of AChE, was 34 +/- 3 min. The reactivation time of HI-6 on soman-inhibited AChE was 6 +/- 2 min. These results indicated that AFM was a useful tool in pharmacology and toxicology, and could reveal time information of the interactions between AChE and its ligands.