Organophosphate compounds are responsible for a large number of accidental and/or suicidal exposures and have been used also for warfare and terrorism. The mechanism of toxicity is by inhibition of cholinesterase. Oximes are the only enzyme reactivators clinically available but clinical experience with oximes is disappointing. There is a gap between laboratory data and clinical impression concerning the efficacy of oxime compounds. Oximes are responsible for thiocholinesteratic activity, a spurious signal caused by interaction between pralidoxime and the thiocholine substrate used for photometric enzyme activity determinations. In a prospective, controlled, non-randomized study performed in anaesthetized miniature pigs, we quantified the extent of pralidoxime-induced cholinesteratic pseudo-activity ex vivo (human blood) and in vivo (minipig) in order to be able to correct values obtained by photometric methods. Plasma cholinesteratic activity using two substrates (acetylthiocholine and butyrylthiocholine) was determined in vitro and in vivo in the presence of pralidoxime. Pralidoxime reacts with the substrate (acetyl- and butyrylthiocholine) used for enzyme activity determinations, producing a spurious signal implying cholinesterase activity (even in the absence of plasma and thus of any enzyme). Cholinesterase activities determined photometrically after pralidoxime therapy can be erroneously high. Although in theory this could mislead clinicians into assuming an efficacious therapy, this is unlikely to occur in vivo under normal pralidoxime dosing conditions. To avoid any ambiguity it is recommended that blood be drawn for enzyme activity determinations prior to reactivator use and no less than 1 h after its administration.