Exposure to the organophosphorus nerve agents such as sarin, soman, cyclosarin, and VX causes acute intoxication by inhibiting acetylcholinesterase (AChE), where the serine residue of the active site can attack the phosphorous atom of the organophosphorus agents to form a strong P-O bond. The purpose of the present study was to evaluate new oxime antidotes to reactivate the inhibited AChE. We have designed and synthesized several new oximes, and have evaluated the substances that differ from the currently used oximes in linker between the two pyridinium rings. The potency of newly synthesized oximes was compared with two currently used AChE reactivators (2-PAM, HI-6). The reactivation potencies of the bis-pyridinium oximes connected with a (CH(2))(n) linker between the two quaternary nitrogen atoms were evaluated with housefly (HF) AChE inhibited by diisopropyl fluorophosphates (DFP) and by paraoxon. The bis-pyridinium oximes showed stronger activity compared with mono-pyridinium oxime, and the magnitude of reactivation potency depended on the length of the methylene linker. The potency order was (CH(2))<(CH(2))(2)<(CH(2))(3)>(CH(2))(4)>(CH(2))(7). A (CH(2))(3) linker was optimal in HF AChE inhibited by either DFP or paraoxon. Thus, bis-pyridinium oxime 5 which has (CH(2))(3) linker showed the highest activity in this series of compounds. Interestingly, 5 was not as active as 2-PAM, showing that the position of the oxime group on the pyridinium ring is also very important for the reactivation potency.