Kinetic and toxicological characteristics of fish (Odontesthes argentinensis) and crab (Callinectes sapidus) cholinesterases as well as methodological conditions to perform reactivation assays using pyridine 2-aldoxime (2-PAM) were established. According to kinetic and eserine sensitivity data, both cholinesterases can be considered as acetylcholinesterases. The concentration of eserine that inhibited 50% of enzyme activity (IC(50)) was estimated as 15.9x10(-8) and 4.6x10(-8) M for crab and fish, respectively. For purified eel acetylcholinesterase (V-S type), it was estimated as 4.2x10(-8) M. 2-PAM showed both to increase non-enzymatic hydrolysis of acetylthiocholine iodide and to inhibit activity of the acetylcholinesterases tested. The IC(50) of 2-PAM for crab acetylcholinesterase (8.2x10(-4) M) was significantly higher than that from O. argentinensis (2.5x10(-4) M) or eel (2.0x10(-4) M) acetylcholinesterase. Enzyme inhibition induced by 2-PAM showed to mask subtle inhibition due to malathion, suggesting that a previous characterization of 2-PAM inhibition must be done before its use in reactivation assays.