The potential use of antioxidant system parameters has gained considerable interest due to their pivotal role of detoxification mechanisms in environmental studies and culture fish point of view. Fish with different ecological needs may have different antioxidant capacity and response to environmental contaminants. Thus, the optimal working conditions and specific enzyme activities (V-max and K-m) of antioxidant system parameters (Superoxide dismutase, SOD; Catalase, CAT; Glutathione peroxidase, GPX; Glutathione reductase, GR and Glutathione S-transferase, GST) and glutathione (GSH) were determined in four commonly cultured freshwater fish species (tilapia; Oreochromis niloticus, carp; Cyprinus carpio, trout; Onchorhynchus mykiss and catfish; Clarias garipienus). Data showed that optimal concentrations of different buffers, pH and specific chemicals for each enzyme and GSH were similar in most cases for all fish species, except a few differences. The highest Vmax and Km values were found in carp for GPX and GST, though these values were the highest in tilapia, catfish and trout for CAT, SOD and GR, respectively. As a conclusion, optimization assays of these parameters in different bioindicator organisms based on their physiological and ecological differences may be useful for the aquatic ecosystem biomonitoring studies and also present fundamental data for utilization in aquaculture. (C) 2015 Elsevier Inc. All rights reserved.