The therapeutic approach of organophosphorus compound (OP) intoxications is to reactivate the inhibited enzyme acetylcholinesterase (AChE). Numerous studies demonstrated a limited efficacy of standard oxime-based reactivators against different nerve agents such as tabun and cyclosarin. This emphasizes research for more effective oximes. In the present study, reactivation kinetics of tabun-, sarin-, cyclosarin-, VX-or paraoxon-ethyl-inhibited human AChE (hAChE) with a homologous series of bis-ortho-pyridiniumaldoximes, Ortho-4 -Ortho-9, was investigated with a robot-assisted setting, allowing determination of second-order reactivation rate constants as well as model calculations. The reactivation constants of Ortho-4 -Ortho-9 resulted in marked differences of affinity and reactivity depending on the OP structure and the linker length of the oximes. In general, the K D values decreased with increasing linker length.Reactivity increased from Ortho-4 to Ortho-6 for PXE-and VX-inhibited hAChE and from Ortho-4 to Ortho-7 for GA-inhibited hAChE and decreased again with Ortho-8 and Ortho-9. In contrast, k r decreased with increasing linker length for sarin-and cyclosarin-inhibited hAChE. In view of the pronounced decrease of K D from Ortho-4 to Ortho-9, the k r2 values increased with all tested OP. Hence, the ratios of K I / K D and of K I / k r2 showed that in almost all cases the affinity of Ortho-N to the native hAChE was higher than to OP-inhibited enzyme. Model calculations indicated that Ortho-6 -Ortho-9 could be superior to obidoxime in reactivating tabun-inhibited hAChE. Finally, these data emphasize the need to develop oximes with a higher selective affinity towards OP-inhibited hAChE in order to minimize possible side effects.