Due to the current controversy about the real effectiveness of the oximes in the treatment of organophosphate poisoning, the reactivation capacity of pralidoxime has been evaluated in vitro on human erythrocyte acetylcholinesterase inhibited by dimethoate. In the in vitro model, a partial recovery of acetylcholinesterase activity was observed with concentrations from 0.066 mM pralidoxime, probably useful enough to prevent death in most cases in vivo. However, much more effectiveness was observed with concentrations up to 0.70 mM pralidoxime. Although pralidoxime should be applied as soon as possible after organophosphate exposure, the application of the antagonist can be useful even 24h after, particularly for organophosphates with biological half-life longer than one day. The protective capacity of pralidoxime after the application was reduced up to 50% in 6h and disappeared almost completely in 24h. Furthermore, the pesticide and its metabolites remained active and were able to inhibit the enzyme as soon as pralidoxime reduced its antagonist capacity. Our results in conjunction with the short half-life of pralidoxime suggest that the maintenance of higher plasmatic concentrations than the currently used should be considered in the management of severe poisoned patients, although adverse effects could be expected.