The influence of several variables such as sample and reagent storage, anticoagulants, reaction temperature, pH, and substrate concentration on whole blood cholinesterase determination was studied. Storage of nondiluted whole blood samples at room temperature or under refrigeration (4 C) was adequate for short-term storage (3 days to 2 weeks). However, freezing would be more appropriate for long-term storage (> or = 1 month), and successive thawing and freezing did not produce any loss of cholinesterase activity. All reagents (2,2'-dithiodipyridine as chromophore and acetylthiocholine and butyrylthiocholine as substrates) were stable for 3 months when frozen. Heparin and ethylenediaminetetraacetic acid were the most suitable anticoagulants for whole blood acetylcholinesterase and butyrylcholinesterase determination, because citrate yielded lower acetylcholinesterase values and fluoride inhibited butyrylcholinesterase. Increases in reaction temperature and pH yielded higher cholinesterase values but also increased nonenzymatic substrate hydrolysis. Higher cholinesterase and nonenzymatic substrate hydrolysis values were obtained as higher substrate concentrations were used.