Brain acetylcholinesterase (AChE; EC 3.1.1.7) from peacock bass (Cichla ocellaris) was characterized and the effect of organophosphorus and carbamate pesticides as well as ions and heavy metals was evaluated. The kinetic parameters K_m and V_max were determined as 0.769mM and 0.189U/mg of protein respectively. Optimal pH and temperature were found to be 8.0 and 45°C. The enzyme retained approximately half of the activity after incubation at 50°C for 30min. Total cholinesterase activity on brain of this species can be ascribed to AChE according to selective inhibitors analysis (neostigmine, eserine and BW284c5 reduced its activity whereas no effect was noticed for Iso-OMPA). Seven pesticides (five organophosphates: dichlorvos, diazinon, chlorpyrifos, temephos, tetraethyl pyrophosphate – TEPP and two carbamates: carbaryl and carbofuran) showed inhibitory effects on C. ocellaris AChE. However, the strongest effect was observed with carbofuran (IC₅₀=0.21μM and K_i=2.57×10⁻³μM). The following ions (1mM) showed to inhibit its activity (decrescent order): Hg²⁺>As³⁺>Cu²⁺>Zn²⁺. EDTA²⁻ did not affect enzyme activity. The present study provides assay conditions and data to suggest this enzyme as in vitro biomarker of organophosphorus and carbamate pesticides in routine environmental screening programs.