Inhibited cholinesterase in tissues of animals exposed to carbamate pesticides is known to reactivate readily, presenting considerable problems in the accurate assessment of cholinesterase activity in these tissues. Decarbamylation of cholinesterase is favored when the tissue samples are diluted and/or are incubated for an extended time. The present study was performed to identify modifications of the commonly used spectrophotometric assay for cholinesterase activity that would minimize spontaneous reactivation of enzyme activity. Those modifications included preincubation of concentrated tissue with concentrated chromogen (i.e., DTNB), dilution to final reaction volume immediately before measurement, and measurement of cholinesterase over a short period of time (5-10 min). The Ellman assay with and without modifications was performed using a microtiter plate reader on tissues from carbaryl-treated rats:undiluted plasma, diluted erythrocytes (1:25), minimally diluted erythrocytes (1:2), diluted brain (1:100), or minimally diluted brain (1:2). The results were compared to cholinesterase activities obtained using a radiometric method which employs minimally diluted tissue and short incubation times. The degree of cholinesterase inhibition for undiluted or minimally diluted tissue assayed by the modified method agreed with those obtained using the radiometric method. Even if the tissues were diluted immediately before assay, however, significant reactivation occurred by the time the first measurements were made by the conventional method. Furthermore, significant spontaneous reactivation may still occur using the modified method if the assay is run for more than 10 min. Use of this modified Ellman method will enable more accurate estimation of in vivo cholinesterase activity in animals treated with carbamates.